Theragnostic particles

ABSTRACT

The present invention provides viral-based nanoparticles for therapeutic and diagnostic use, and methods for making and using the nanoparticles. Specifically, such nanoparticles comprise decoration-competent viral particles shells such as expanded capsids of phages, stabilized with engineered decoration proteins that have been linked to one or more compounds not naturally occurring on a wild type viral capsid.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of, and claims priority to,U.S. patent application Ser. No. 14/652,645, filed Jun. 16, 2015, nowU.S. Pat. No. 9,765,122, which is the U.S. national phase applicationfiled under 35 U.S.C. § 371 claiming priority to International PatentApplication No. PCT/US2014/012206, filed Jan. 20, 2014, which claimspriority under 35 U.S.C. § 119(e) to U.S. Provisional Patent ApplicationNo. 61/754,458, filed Jan. 18, 2013, all of which are herebyincorporated by reference in their entireties herein.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with U.S. Government support under grant numberMCB-0648617 and grant number MCB-1550993, awarded by the NationalScience Foundation. The U.S. Government has certain rights in theinvention.

BACKGROUND OF THE INVENTION

Viral-based systems can be modified to present a high density of ligandsin a defined symmetric pattern; this type of display can increase theavidity of binding to target biomolecules. For example, bacteriophagelambda has been used for phage display applications through peptidefusions with either the major tail protein (gpV) or the gpD decorationprotein (Maruyama et al., 1994; Mikawa et al., 1996). Studies have shownthat gpD may be modified at either the N- or C-terminus to presentpeptides at the capsid surface for phage display applications. However,all current phage based display systems are limited by the requiredconstruction of decoration or major capsid protein fusion constructswithin the context of an infectious viral particle, while the constructshave been constructed in vivo, thus limiting these systems to peptideand protein fusion constructs expressed within infected cells in thecontext of an infectious virus. As a result, the stoichiometry of thefusion proteins cannot be controlled on the resulting infectious viralparticles, the modified constructs are limited to peptide and proteindisplay ligands, and the fusion proteins are limited to fusions at theN- or C-terminus of the decoration protein.

SUMMARY OF THE INVENTION

In one aspect, the present invention provides theragnostic particles,comprising a plurality of engineered decoration proteins bound to anouter surface of decoration competent viral particle shell, wherein theengineered decoration proteins comprise decoration proteins linked toone or more compounds not naturally occurring on a wild type viralcapsid, and wherein the one or more compounds have at least one featureselected from the group consisting of:

(a) the one or more compounds are non-proteinaceous compounds;

(b) the one or more compounds are present on the theragnostic particlein a defined ratio relative to the engineered decoration protein;

(c) the one or more compounds comprise two or more different compounds,wherein the two or more different compounds are present on thetheragnostic particle in a defined ratio relative to each other; and

(d) the one or more compounds are linked to the engineered decorationprotein at a site on the engineered decoration protein other than theN-terminus or the C-terminus.

In another aspect, the invention provides pharmaceutical composition,comprising the theragnostic particles of the invention and apharmaceutically acceptable carrier.

In a further aspect, the invention provides an isolated recombinantprotein comprising the amino acid sequence of SEQ ID NO: 10 (gpD(S42C)),as well as gpD S42C linked to one or more compounds of interest. Theinvention also provide isolated nucleic acids encoding the isolatedprotein, recombinant expression vectors comprising the isolated nucleicacids, and isolated host cells comprising the recombinant expressionvectors of the invention.

In another aspect, the invention provides an in vitro method forpreparing a theragnostic particle, comprising decorating a decorationcompetent viral particle shell in vitro with a defined amount ofengineered decoration proteins linked to one or more compounds, whereinthe engineered decoration proteins stabilize the decoration competentviral particle shell to produce a theragnostic particle.

SUMMARY OF THE FIGURES

FIG. 1. Phage Lambda Assembly Pathway. A multi-genome concatemer isdepicted, which serves as the preferred DNA packaging substrate in vivo.Details of the packaging pathway are provided in the Example 1.

FIG. 2. Expansion of the Lambda Procapsid in Vitro. Panel A. Magnesiumand Urea Stabilize the Procapsid and Expanded Capsid Shells.Respectively. Procapsids were expanded in 2.5 molar urea, which affordsthe expanded capsid shell (Urea). The expanded shells were then bufferexchanged into either high magnesium (15 mM, TMB) or low magnesium (1mM, TMSO) buffer, as indicated. The migration of the procapsid (●) andexpanded capsid (●) in the agarose gel is indicated at right of the gel.Panel B. Urea-Triggered Expansion is Strongly Temperature Dependent.Procapsids were incubated in 2.5 molar urea for 15 minutes at theindicated temperature and then analyzed by 0.8% agarose gel. Panel C.Urea-Triggered Expansion is Inhibited by Salt. Procapsids were expandedas described in Materials and Methods except that NaCl was added to thereaction mixture as indicated. Panel D. Procapsid Expansion is FullyReversible. Procapsids were expanded in 2.5 molar urea for 15 minutes at4° C. (Urea) and then buffer-exchanged into TMB buffer. The identicalsample was again expanded and buffer exchanged as indicated.

FIG. 3. Thermodynamic Characterization of Urea-Triggered ProcapsidExpansion. Panel A. Procapsids in the absence (◯) or in the presence of3 mM (●) or 10 mM (⋄) MgCl₂ were incubated on ice in the presence ofincreasing concentrations of urea, as indicated, and the fraction ofexpanded capsids was quantified by gel assay. Each data point representsthe average of at least three separate measurements with standarddeviations indicated with bars. The solid line represents the best fitof the data to equation 1. Panel B. The data presented in Panel A wasanalyzed according to equation 2 to afford the concentration of urearequired to expand half of the procapsid shells ([urea]_(1/2)). Panel C.The free energy of capsid expansion (●) and the denaturant m values (◯)derived from Panel A are plotted as a function of MgCl₂.

FIG. 4. Magnesium Drives Contraction of the Expanded Capsid to theProcapsid State. MgCl₂ at the indicated concentration was added toexpanded capsids in the absence (◯) or presence (●) of 2.5 molar urea asdescribed in Materials and Methods. The fraction of contracted capsidswas quantified by agarose gel assay. Each data point represents theaverage of at least three separate measurements with standard deviationsindicated with bars. The solid lines represent the best fit of the dataaccording to Equation 3.

FIG. 5. Urea-Expanded Capsids are Biologically Functional Panel A.Expanded Capsids Bind the gpD Decoration Protein. Procapsids andexpanded capsids (in the absence of urea) were incubated with gpD asdescribed in Materials and Methods and the products were analyzed by0.8% agarose gel. Note that the gpD-decorated shell migrates faster thanboth the procapsid and the expanded capsid shells. Panel B. The gpDProtein Stabilizes the Expanded Capsid Shell. Micrographs of negativelystained procapsids (i), expanded capsids (ii), expanded capsids thathave been re-contracted with 15 mM MgCl₂ (iii), and gpD-decoratedexpanded shells (iv). Note that the expanded shells are fragile anddeteriorate during preparation for EM analysis. Expanded capsids thathave been re-contracted with magnesium or stabilized with gpD arestructurally sound. Panel C. Expanded Capsids are Biologically Active.Procapsids and GpD-decorated expanded procapsids (Capsids) were used inan in vitro DNA packaging assay. The data represent the average of threeseparate experiments with standard deviations indicated with bars.Inset. DNAase resistant (packaged) viral DNA was analyzed by gel assay.Note that only full-length genomic DNA (48.5 kb, arrow) is renderedDNAase resistant. This indicates that both capsid preparations packageDNA in a processive manner.

FIG. 6. Model for Reversible Procapsid Expansion and gpD Addition. UpperPathway. Procapsid expansion is triggered by DNA packaging in vivo. Mg²⁺strongly stabilizes the procapsid conformation. Packaged DNA binds Mg²⁺at the interior procapsid surface, lowering the free energy required forthe expansion transition. GpD trimer spikes assemble at hydrophobicpatches exposed at the icosahedral three-fold axes of the expanded shelllattice and stabilize the particle. Lower Pathway. Procapsid expansionis triggered by urea in vitro. Mg²⁺ strongly stabilizes the procapsidconformation and higher concentrations of urea are required to triggerthe transition. GpD trimer spikes assemble at hydrophobic patchesexposed at the icosahedral three-fold axes of the expanded shelllattice. The decorated particles are structurally robust andbiologically active.

FIG. 7. Bacteriophage lambda capsid in vivo (Panel A) and in vitro(Panel B) assembly. At full saturation, 420 copies of gpD decorate thecapsid surface, which is composed of 415 copies of the major capsidprotein (gpE). Details described in example 2.

FIG. 8. The gpD Trimer Spike and gnD Constructs Used in this Study.Panel A. Side view of the gpD-WT trimer. The gpD trimer is shown incartoon representation with each subunit colored a different shade ofblue and with serine 42 depicted as red spheres. Panel B. The gpDconstructs used in this study.

FIG. 9. GFP-Tagged gpD Adds to the Lambda Capsid in Vitro. Expandedcapsids were incubated with an increasing ratio of gpD-wt:gpD-GFP asindicated. The decoration mixture was fractionated by agarose gelelectrophoresis and the gel was stained with either Coomassie blue(Panel A) or visualized with UV light (Panel B) as described inMaterials and Methods. Note that unincorporated H6-gpD-GFP also appearson the agarose gel. Panel C. Unreacted gpD-WT and gpD-GFP was removedfrom the decoration reaction mixture using Amicon Ultra-0.5centrifugation filters and the decorated capsids were analyzed bySDS-PAGE.

FIG. 10. Expanded capsids can be decorated with varying ratios of gpDand gpD(S42C::sMannose) (sMannose). Panel A. Decorated capsids wereseparated from excess gpD and sMannose on an agarose gel and stainedwith Coomassie. Panel B. Decorated capsids were separated fromunincorporated gpD and sMannose by buffer exchange using AmiconUltra-0.5 centrifugation filters and the protein content of theparticles were analyzed by SDS-PAGE.

FIG. 11. Lambda Capsids Can be Decorated with Multiple Ligands in aDefined Ratio. Panel d. Expanded capsids can be decorated with varyingratios of gpD(S42C), sMannose, and gpD-GFP. Decorated capsids wereseparated from excess gpD and modified gpD on an agarose gel and stainedwith Coomassie. The migration of the decorated capsids on an agarose gelwas unique for each variant. Panel C. When illuminated by UV light,GFP-decorated capsids fluoresce. Panel C. Decorated capsids wereseparated from unincorporated gpD, sMannose and gpD-GFP by bufferexchange using Amicon Ultra-0.5 centrifugation filters and the particleswere analyzed by SDS-PAGE. Panel D. To assess the ability of decoratedcapsids to bind to Concanavalin A (Con A), a mannose-specific lectin,agglutination assays were performed. Only those decorated with sMannosebound specifically to Con A.

FIG. 12. Electron micrographs of decorated capsids. Details described inexample 2. Scale bar, 50 nm.

FIG. 13. Capsids decorated with modified gpD retain DNA packagingactivity. Panel A. Capsids fully decorated with sMannose are fullycompetent, but those decorated with 100% gpD-GFP are compromised whenfull-length lambda is used as the packaging substrate. Panel B. Capsidsdecorated with up to 50% gpD-GFP are fully competent for packaging.

DETAILED DESCRIPTION OF THE INVENTION

It must be noted that, as used in the specification, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise.

Any terms not directly defined herein shall be understood to have themeanings commonly associated with them as understood within the art ofthe present disclosure. Certain terms are discussed herein to provideadditional guidance to the practitioner in describing the compositions,devices, methods, and the like, of embodiments of the presentdisclosure, and how to make or use them. It will be appreciated that thesame thing can be said in more than one way. Consequently, alternativelanguage and synonyms can be used for any one or more of the termsdiscussed herein. No significance is to be placed upon whether or not aterm is elaborated or discussed herein. Some synonyms or substitutablemethods, materials and the like are provided. Recital of one or a fewsynonyms or equivalents does not exclude use of other synonyms orequivalents, unless it is explicitly stated. Use of examples, includingexamples of terms, is for illustrative purposes only and does not limitthe scope and meaning of the embodiments of the present disclosureherein.

As used herein, “about” means+/−10% of the recited value.

All embodiments disclosed herein can be used in combination, unless thecontext clearly indicates otherwise. Unless the context clearly requiresotherwise, throughout the description and the claims, the words‘comprise’, ‘comprising’, and the like are to be construed in aninclusive sense, as opposed to an exclusive or exhaustive sense; that isto say, in the sense of “including, but not limited to”. Words using thesingular or plural number also include the plural or singular number,respectively. Additionally, the words “herein,” “above” and “below” andwords of similar import, when used in this application, shall refer tothis application as a whole and not to any particular portions of thisapplication.

The following description provides specific details for a thoroughunderstanding of, and enabling description for, embodiments of thedisclosure. However, one skilled in the art will understand that thedisclosure may be practiced without these details. In other instances,well-known structures and functions have not been shown or described indetail to avoid unnecessarily obscuring the description of theembodiments of the disclosure. Persons skilled in the relevant art canappreciate that many modifications and variations are possible in lightof the embodiment teachings. Accordingly, the disclosure is intended tobe illustrative, but not limiting, of the scope of invention.

In a first aspect the present invention provides an in vitro method forpreparing a theragnostic particle, comprising decorating a decorationcompetent viral particle shell in vitro with a defined amount ofengineered decoration proteins linked to one or more compounds, whereinthe engineered decoration proteins stabilize the decoration competentviral particle shell to produce a theragnostic particle.

The present invention overcomes the shortcomings of viral particlesystems in the art, which did not permit capsid decoration with adefined amount of engineered decoration proteins linked to compounds ofinterest. The methods of the present invention permit construction oftheragnostic (i.e.: can be used for therapeutic or diagnostic use)particles decorated with defined amounts of engineered decorationprotein, and where the engineered decoration proteins are linked to anycompound or multiple compounds of interest including non-proteinaceouscompounds, which can themselves be added in defined amounts to theresulting viral particle. Further, the engineered decoration proteins ofthe invention can be modified at any suitable position on the proteinthat does not interfere with binding to the expanded viral particleshell. As such, the theragnostic particles made by the methods of theinvention can be used for any suitable therapeutic or diagnosticpurpose, as described in more detail herein. In one embodiment, theexpanded viral particle shells are decorated with a stoichiometric ratioof engineered decoration proteins.

As used herein, the “viral particle” is a protein shell of a virus(capsid) or virus-like particle. For use in the present invention, theviral particles are based on capsids of double stranded DNA viruses thatnormally package their genome into preassembled procapsid shells.Packaging in these viruses triggers a major re-orientation and expansionof the proteins assembled into the procapsid shells, yielding the maturecapsid shell that is subsequently stabilized with the addition ofdecoration proteins. Exemplary such double stranded DNA viruses include,but are not limited to bacteriophages lambda, T4, 21, L, P4, herpesvirusand adenoviruses.

The viral particle can be an isolated capsid or may be produced by anysuitable means, such as recombinant expression of the structuralproteins and subsequent viral particle assembly using the recombinantlyexpressed structural proteins in vitro; in the latter case the particlesare known as virus-like particles (VLP). In one embodiment, described inmore detail in the examples that follow, vectors can be designed toexpress all proteins required to assemble a functional capsid in a hostcell, such as E. coli, and the particle can then be purified. The viralparticle may comprise or consist of wild type or mutated capsidstructural proteins, so long as the mutated structural proteins can formthe engineered viral particle (e.g., functional mutant structuralproteins). The viral particles may be isolated and stored orpreassembled and stored, or may be isolated or assembled at the time ofcarrying out the methods of the invention. Assembly or isolation can becarried out by any suitable means, which are well within the level ofskill in the art based on the disclosure herein. Exemplary isolation andassembly techniques are provided in the examples that follow.

In one embodiment, a virus-like particle is produced by in vitroassembly of isolated capsid structural proteins. The capsid structuralproteins to be used will depend on the virus that the viral particle isbased on. Capsid structural proteins for each of bacteriophages lambda,T4, L, P22, 21, and P4 and for the eukaryotic adenovirus andherpesviruses are well known to those of skill in the art, as aremethods for their recombinant production or isolation. In onenon-limiting embodiment, a lambda phage viral particle is used. In thisembodiment, the structural proteins may be portal protein gpB (SEQID: 1) and major capsid protein gpE (SEQ ID: 2), and the minor capsidprotein gpC (SEQ ID: 3), or the scaffolding protein contained therein(SEQ ID NO: 36; residues 309 to 439 of SEQ ID NO:3), or functionalmutants thereof.

In another embodiment, a bacteriophage T4 viral particle is used; inthis embodiment, the structural proteins are the T4 structural proteins,or functional mutants thereof. In a further embodiment, a bacteriophageL viral particle is used; in this embodiment, the structural proteinsare the bacteriophage L structural proteins, or functional mutantsthereof. In another embodiment, a bacteriophage P22 viral particle isused; in this embodiment, the structural proteins are the bacteriophagep22 structural proteins, or functional mutants thereof. In anotherembodiment, a bacteriophage 21 viral particle is used; in thisembodiment, the structural proteins are the bacteriophage 21 structuralproteins, or functional mutants thereof. In a further embodiment, abacteriophage P4 viral particle is used; in this embodiment, thestructural proteins are the bacteriophage P4 structural proteins, orfunctional mutants thereof. The structural proteins and their amino acidsequences for each of these viral structural proteins known to those ofskill in the art.

The viral particle is “decoration competent” in that the particle iseither (a) prepared or isolated in a form that can add decorationprotein to the shell without further manipulation, (b) contacted with anexpansion agent in vitro under conditions and for a time suitable toproduce an expanded viral particle shell, that will be stabilized in theexpanded state by decoration with the decoration protein, or (c)contacted with an agent in vitro under conditions and for a timesuitable to produce a shell that has undergone a conformational changesuch that it will be stabilized in that conformation state by decorationwith the decoration protein. Packaging in phages lambda, T4, 21, P22,and L, DNA triggers major conformational changes that alter the shellmorphology and cause shell expansion. The herpesviruses similarlyundergo major conformational changes with DNA packaging that alter theshell morphology but they are not typically described as expanding.Purification affords a mixture of pre-change and post-change species inmost cases.

Thus, in one embodiment, the viral particle shell has under extensiveconformational change that renders the shell competent for decorationwith the decoration protein. All procapsids undergo a major structuralre-organization of the shell as a result of DNA packaging during theviral particle maturation process. Decoration of the shell with thedecoration protein stabilizes this embodiment of a decoration competentviral particle shell.

In another embodiment, the methods involve expanding the viral particleby contacting it with an expansion agent in vitro under conditions andfor a time suitable to produce an expanded viral particle shell. Anysuitable expansion agent that can expand a specific viral particle canbe used, including but not limited to chaotropic agents (including butnot limited to urea), pH changes, heating, etc. The methods do notrequire any specific amount of expansion of the viral particle; theexpansion may be similar to the expansion seen upon genome packagingduring the normal viral life cycle. Any suitable expansion agent can beused, as deemed most appropriate in light of the specific of theengineered viral particle and all other relevant factors. In oneembodiment, a chaotropic agent such as urea is used. In one non-limitingexample, when bacteriophage lambda viral particles are used, urea can beused as the chaotropic agent; in this embodiment, the resulting viralparticle shell can optionally be contracted and expanded repeatedly ifdesired. In a further embodiment using bacteriophage lambda viralparticles, expansion of the cell is done at about 4° C. In otherembodiments, the expansion agent can be heat (range of 4° C. to 50° C.),pH (pH range of 3-9), or other expansion agent. Based on the teachingsherein and the specific viral particle being used, those of skill in theart will be able to determine the most appropriate expansion agent for agiven use. Similarly, conditions and times suitable to produce theexpanded virus shell can be determined by those of skill in the artbased on the teachings herein and in light of the specific viralparticle being used.

In another embodiment, the methods, agents, and techniques described forparticle expansion are applied to driving the conformational change ofthe shell required to afford a decoration competent particle shell, inthose viral capsids that do not undergo particle expansion.

The methods comprise decorating the decoration competent viral particleshells in vitro with engineered decoration proteins. As used herein“decorating” means binding a plurality of decoration proteins to theviral particle shells, such that the decoration proteins stabilize thedecoration competent viral particle shell. Since the methods are carriedout in vitro, the decoration proteins can be added stochiometrically, inany desired amount to the decoration competent viral particle shells. Asis known by those of skill in the art, a variety of double stranded DNAviruses possess decoration proteins that stabilize procapsid shells toproduce the mature capsid shell. Non-limiting examples of suchdecoration proteins include, but are not limited to, the lambda gpDdecoration protein, T4 head outer capsid (Hoc) protein, T4 small outercapsid (Soc) protein, Shp protein of phage 21, the Dec protein of phageL, and the Psu protein of phage P4, and protein IX of adenoviruses. Theplurality of decoration proteins that are added depend on the viralparticle used, the modifications made to the decoration protein, and theintended use of the viral particles, but include enough decorationproteins to bind to at least 80% of the binding sites for the decorationprotein that are present on the expanded viral particle shell (e.g.: onthe structural proteins defining the shells. In various furtherembodiments, the engineered decoration proteins bind to at least 85%,90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or to all binding sites for thedecoration protein that are present on the expanded viral particleshell.

As will be understood by those of skill in the art, the choice ofdecoration protein will depend on the viral particle/structural proteinsbeing used. Thus, if the viral particle is lambda phage based, lambdaphage structural proteins (or functional mutants thereof) and theengineered lambda decoration protein (engineered gpD protein) will beused. The wild type gpD amino acid sequence is shown in SEQ ID: 4.Similarly, if the viral particle is bacteriophage T4 then T4 structuralproteins (or functional mutants thereof) and the engineered T4decoration protein (engineered Hoc or Soc protein) will be used. Thewild type Hoc amino acid sequence is shown in SEQ ID: 5; The wild typeSoc amino acid sequence is shown in SEQ ID: 6. If the viral particle isbacteriophage 21, then bacteriophage 21 structural proteins (orfunctional mutants thereof) and the engineered bacteriophage 21decoration protein (Shp protein) will be used. The wild type Shp aminoacid sequence is shown in SEQ ID: 7. If the viral particle isbacteriophage L, then bacteriophage L structural proteins (or functionalmutants thereof) and the engineered bacteriophage L decoration protein(Dec protein) will be used. The wild type Dec amino acid sequence isshown in SEQ ID: 8. If the viral particle is bacteriophage P22, thenbacteriophage P22 structural proteins (or functional mutants thereof)and the engineered bacteriophage L decoration protein (Dec protein) willbe used, since P22 does not make a decoration protein in vivo, but canbe decorated with Dec from phage L in vitro. If the viral particle isbacteriophage P4, then bacteriophage P4 structural proteins (orfunctional mutants thereof) and the engineered bacteriophage P4decoration protein (Psu protein) will be used. The wild type Psu aminoacid sequence is shown in SEQ ID: 9.

The decoration proteins for use in the methods of the invention are“engineered” in that they are not the wild type proteins, but have beenmodified and are linked to one or more compounds of interest. Since themethods of the present invention can be carried out in vitro, thedecoration proteins can be modified in any desirable way, providing aunprecedented increase in the functionality that can be displayed on thesurface of the resulting particles compared to prior art methods. In oneembodiment, the decoration proteins have an altered amino acid sequencefrom wild type, such as an insertion, deletion, one or moresubstitutions, etc. Non-natural amino acid residues can be incorporatedinto the engineered decoration proteins as suitable for a given purpose.Any suitable modifications can be made to the decoration proteins solong as the resulting engineered decoration protein can bind to theviral particle shell and stabilize it.

In one non-limiting embodiment, any solvent accessible residue on ashell bound decoration protein can be modified as desired, such as tofacilitate linkage to one or more compounds of interest. For example,residues in the shell-bound gpD trimer spike that are exposed to solventinclude, but are not limited to Thr2, Ser3, Lys4, Glu5, Thr6, Phe7,Thr8, His9, Tyr10, Gln11, Pro12, Gln13, Gly14, Asn15, Ser16; Gly26,Gly27, Leu28, Ser29, Ala30, Lys31, Ala32; Asp41, Thr42, Ser43, Ser44,Arg45, Lys46; Asp51, Gly52, Thr53, Thr54, Asp55; Asp67, Gln68, Thr69,Ser70, Thr71, Thr72; Arg82, Tyr83, Glu84, Asp85; Glu90, Ala91, Ala92,Ser93, Asp94, Glu95, Thr96, Lys97, Lys98, Arg99, Thr100: the N- andC-terminal residues of the protein. In one non-limiting embodiment, oneor more residue may be substituted with a Cys residue (including but notlimited to an S42C substitution), to facilitate binding of othercompounds to the decoration proteins. In another embodiment, anon-natural amino acid, including but not limited to azidohomoalanine(Aha) can be incorporated using genetic manipulation of the gene toallow alternate means for chemical modification of the decorationprotein, including “click” chemistry.

Note that the N-terminal methionine shown for gpD in SEQ ID NO 4 isgenerally excluded from the mature protein by cell degradation. Forpurposes of this application, the numbering for gpD excludes the(optional) N-terminal methionine. Thus, for example, Ser42, representsthe 42 amino acid in the mature gpD sequence (deleted for the N-terminalmethionine), but is the 43^(rd) amino acid in the sequence if theN-terminal methionine is counted. The numbering system follows thatreported in the crystal structure of gpD (Yang et al, (2000) Nat.Struct. Biol. vol 7, pp. 230) which does not included the N-terminalmethonine encoded by the gene sequence.

The engineered decoration proteins comprise engineered decorationproteins linked to one or more compound. In another embodiment, theengineered decoration proteins comprise engineered decoration proteinslinked to one or more compound selected from the group consisting ofnucleic acids, lipids, carbohydrates, polypeptides, polymers, organicmolecules, inorganic molecules, or combinations thereof. In a furtherembodiment, the engineered decoration proteins comprise engineereddecoration proteins linked to one or more non-proteinaceous compound,such as nucleic acids, lipids, carbohydrates, polymers, organicmolecules, inorganic molecules (e.g. magnetic beads and quantum dots,among others), or combinations thereof. In all of these embodiments, thelinkage can be at any suitable position on the decoration protein, whichis not possible using previously known methods (which are limited toengineered fusion proteins, such that linkage is at the N- of C-terminusof the decoration protein). In various embodiments, the linkage can beat the N- or C-terminus of the decoration protein, or at any suitableinternal residue of the decoration protein (e.g., other than theN-terminal or C-terminal residue of the decoration protein). Anysuitable techniques can be used to link the one or more compounds to thedecoration proteins, such techniques are well within the level of thoseof skill in the art based on the teachings herein. As will be understoodby those of skill in the art, any suitable combination of decorationproteins can be used, as appropriate for a given expanded viralparticle.

In further embodiments, the one or more compounds may be any suitablecompound for the theragnostic particles of the invention, including butnot limited to therapeutic compounds, diagnostic compounds, adjuvants,antigens, antibodies, etc.

The decorating comprises contacting the decoration competent viralparticle shells in vitro with the engineered decoration proteins for atime and under conditions suitable to bind the decoration proteins tothe viral particle shells. The range of viral particle decoration islimited by the initial concentrations of the engineered decorationproteins thereby allowing tunability and precise decorating of the viralparticle shell with desired amounts and ratios of the one or morecompounds that is not possible using prior methods. Specific conditionswill depend on the viral particle-decoration protein used, the nature ofthe modification to the engineered decoration protein, and all otherrelevant factors. It is well within the level of those of skill in theart to determine appropriate incubation conditions, based on theteachings herein. Exemplary conditions are provided in the examples thatfollow. In one exemplary and non-limiting embodiment in which lambdaphage viral particles are used with engineered gpD decoration proteins,the contacting is carried out at about room temperature to 37° C.

In all of these embodiments, the one or more compounds linked to thedecoration proteins may comprise two or more different compounds. Inthis further embodiment the two or more different compounds can bepresent on the decoration competent particle shell in a defined ratiorelative to each other. For example, the decoration proteins maycomprise two sets of decoration proteins, a first set linked to compound1 and a second set linked to compound 2. Since the methods of theinvention are carried out in vitro, the decorating can comprisecontacting the expanded viral particles with a ratio of the first andsecond sets of decoration proteins that affords the desiredstoichiometric ratio of the two different compounds on the theragnosticparticle.

In one non-limiting embodiment, the decoration proteins arebacteriophage lambda gpD proteins, or functional mutants thereof. Thelambda gpD decoration protein is a monomer in solution but adds toexpanded viral particles as a trimer spike to each of the 140icosahedral three-fold axes (formed by the structural proteins) on theexpanded viral particle surface. The N-terminus of each gpD proteininteracts with the capsid shell to provide stabilizing contacts requiredfor shell integrity, while the C-termini exit the gpD trimer spikeproximate to the shell surface. Thus, prior art methods that are limitedto linking compounds in vivo to the N- or C-termini of gpD may hindergpD trimer assembly and interfere with its ability to stabilize thecapsid. Further, prior work has been limited to N-terminal or C-terminalpeptide and protein fusion proteins that are used to decorate infectiousphage in vivo. In one embodiment, the engineered gpD proteins comprise aS42C substitution (SEQ ID NO: 10), which can be the sole decorationprotein or can be combined with wild type gpD decoration proteins, orother gpD functional mutants.

The resulting theragnostic particles can be devoid of any packagedmaterial (“empty’) within the particle, or may include any desired cargomaterial packaged on the interior of the particle. For example, DNA isefficiently packaged into the decorated capsids and can be modified tocarry specific genes of interest.

In a second aspect, the present invention provides theragnosticparticles, comprising a plurality of engineered decoration proteinsbound to an outer surface of a decoration competent viral particleshell, wherein the engineered decoration proteins comprise decorationproteins linked to one or more compound not naturally occurring on awild type viral capsid, and wherein the one or more compounds have atleast one feature selected from the group consisting of:

(a) the one or more compounds are non-proteinaceous compounds;

(b) the one or more compounds are present on the theragnostic particlein a defined ratio relative to the engineered decoration protein;

(c) the one or more compounds comprise two or more different compounds,wherein the two or more different compounds are present on thetheragnostic particle in a defined ratio relative to each other; and

(d) the one or more compounds are linked to the engineered decorationprotein at a site on the engineered decoration protein other than theN-terminus or the C-terminus.

All terms used in the first aspect of the invention have the samemeaning when referred to in other aspects of the invention. Thus, thedecoration competent viral particle shell may be either an expandedviral particle that is stabilized in the expanded state by thedecoration proteins, or (b) a viral particle shell that has underextensive conformational change that allows decoration by the decorationprotein.

The compositions of the invention overcome the shortcomings of viralparticle systems in the art. The theragnostic particles of the inventioncomprise stoichiometric amounts of engineered decoration protein, andthe engineered decoration proteins can be linked to any compound ormultiple compounds of interest including non-proteinaceuous compounds,which can themselves be added stochiometrically. Further, the engineereddecoration proteins can be modified at any suitable position on theprotein that does not interfere with binding to the viral particleshell. As such, the theragnostic particles can be used for any suitabletherapeutic or diagnostic purpose, as described in more detail herein.The ability to decorate viral particles with engineered decorationproteins provides an attractive approach to develop “designer”nanoparticles of defined composition and multi-partite, symmetricpresentation.

In one embodiment, the theragnostic particle comprises a lambda phageexpanded viral particle shell is used. In this embodiment, thestructural proteins may be protease gpC/scaffolding protein gpNu3 (SEQID NO: 3) or the scaffolding protein (SEQ ID NO: 36), portal protein gpB(SEQ ID NO: 1) and major capsid protein gpE (SEQ ID NO: 2), orfunctional mutants thereof.

In one non-limiting embodiment, a lambda phage viral particle is used.In this embodiment, the structural proteins may be portal protein gpB(SEQ ID: 1) and major capsid protein gpE (SEQ ID: 2), and the minorcapsid protein gpC (SEQ ID: 3), or the scaffolding protein containedtherein (SEQ ID NO: 36), or functional mutants thereof.

In another embodiment, a bacteriophage T4 viral particle is used; inthis embodiment, the structural proteins are the T4 structural proteins,or functional mutants thereof. In a further embodiment, a bacteriophageL viral particle is used; in this embodiment, the structural proteinsare the bacteriophage L structural proteins, or functional mutantsthereof. In another embodiment, a bacteriophage 21 viral particle isused; in this embodiment, the structural proteins are the bacteriophage21 structural proteins, or functional mutants thereof. In anotherembodiment, a bacteriophage 22 viral particle is used; in thisembodiment, the structural proteins are the bacteriophage 22 structuralproteins functional mutants thereof. In a further embodiment, abacteriophage P4 viral particle is used; in this embodiment, thestructural proteins are the bacteriophage P4 structural proteins, orfunctional mutants thereof. The structural proteins and their amino acidsequences for each of these viral structural proteins are known to thoseof skill in the art.

Non-limiting examples of decoration proteins include, but are notlimited to, the lambda gpD decoration protein (SEQ ID NO:4), T4 headouter capsid (Hoc) protein (SEQ ID NO:5), T4 small outer capsid (Soc)protein (SEQ ID NO:6), Shp protein of phage 21 (SEQ ID NO:7), the Decprotein of phage L (SEQ ID NO:8), and the Psu protein of phage P4 (SEQID NO:9).

The plurality of decoration proteins present on the particle will dependon the viral particle used, the modifications made to the decorationprotein, and the intended use of the viral particles, but include enoughdecoration proteins to bind to at least 80% of the binding sites for thedecoration protein that are present on the expanded viral particle shell(e.g.: on the structural proteins defining the shells). In variousfurther embodiments, the engineered decoration proteins bind to at least85%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or to all binding sites forthe decoration protein that are present on the expanded viral particleshell.

As will be understood by those of skill in the art, the choice ofdecoration proteins will depend on the viral particle shell/structuralproteins being used. Thus, if the viral particle is lambda phage-based,lambda phage structural proteins (or functional mutants thereof) and theengineered lambda decoration protein (engineered gpD protein) will beused. Similarly, if the viral particle is bacteriophage T4 then T4structural proteins (or functional mutants thereof) and the engineeredT4 decoration protein (engineered Hoc or Soc protein) will be used. Ifthe viral particle is bacteriophage 21, then bacteriophage 21 structuralproteins (or functional mutants thereof) and the engineeredbacteriophage 21 decoration protein (shp protein) will be used. If theviral particle is bacteriophage P22, then bacteriophage 22 structuralproteins (or functional mutants thereof) and the engineeredbacteriophage L decoration protein (dec protein) will be used. If theviral particle is bacteriophage P4, then bacteriophage P4 structuralproteins (or functional mutants thereof) and the engineeredbacteriophage P4 decoration protein (psu protein) will be used.

The decoration proteins for use in the methods of the invention are“engineered” in that they are not the wild type proteins, but have beenlinked to one or more compound not naturally occurring on a wild typeviral capsid to provide added functionality. The modifications compriseone or more of

(a) the one or more compounds being non-proteinaceous compounds;

(b) the one or more compounds are present on the theragnostic particlein a defined ratio relative to the engineered decoration protein;

(c) the one or more compounds comprise two or more different compounds,wherein the two or more different compounds are present on thetheragnostic particle in a defined ratio relative to each other; and

(d) the one or more compounds are linked to the engineered decorationprotein at a site on the engineered decoration protein other than theN-terminus or the C-terminus

The decoration proteins can be modified in any desirable way asdisclosed herein, providing a unprecedented increase in thefunctionality that can be displayed on the surface of the resultingparticles compared to prior art methods. In one embodiment, thedecoration proteins have an altered amino acid sequence from wild type,such as an insertion, deletion, one or more substitutions, etc. Anysuitable modifications can be made to the decoration proteins so long asthe resulting engineered decoration protein can bind to the viralparticle shell and stabilize it. In one non-limiting embodiment, anysolvent accessible residue on a shell bound decoration protein can bemodified as desired, such as to facilitate linkage to one or morecompounds of interest.

The engineered decoration proteins comprise engineered decorationproteins linked to one or more compound of interest. In one embodiment,the one or more compounds are selected from the group consisting ofnucleic acids, lipids, carbohydrates, polypeptides, polymers, organicmolecules, inorganic molecules, or combinations thereof. In a furtherembodiment, the engineered decoration proteins comprise engineereddecoration proteins linked to one or more non-proteinaceous compound,such as nucleic acids, lipids, carbohydrates, polymers, organicmolecules, inorganic molecules (e.g. magnetic beads and quantum dots,among others), or combinations thereof. In all of these embodiments, thelinkage can be at any suitable position on the decoration protein, whichis not possible using previously known methods (which are limited toengineered fusion proteins, such that linkage is at the N- of C-terminusof the decoration protein). In various embodiments, the linkage can beat the N- or C-terminus of the decoration protein, or at any suitableinternal residue of the decoration protein (e.g., other than theN-terminal or C-terminal residue of the decoration protein). The one ormore compounds can be linked to the decoration proteins using anysuitable linkage; determining an appropriate linkage based on thespecifics of the one or more compounds and the decoration protein arewell within the level of those of skill in the art based on theteachings herein. A s will be understood by those of skill in the art,any suitable combination of decoration proteins can be used, asappropriate for a given expanded viral particle.

In further embodiments, the one or more compounds may be any suitablecompound for the theragnostic particles of the invention, including butnot limited to therapeutic compounds, diagnostic compounds, adjuvants,antigens, antibodies, etc.

The one or more compounds linked to the decoration proteins may comprisetwo or more different compounds. In this embodiment the two or moredifferent compounds can be present on the viral particle shell in adefined ratio relative to each other. For example, the decorationproteins may comprise two sets of decoration proteins, a first setlinked to compound 1 and a second set linked to compound 2. Since themethods of the invention are carried out in vitro, the decorating cancomprise contacting the decoration competent viral particles with aratio of the first and second sets of decoration proteins that reflectsthe desired stoichiometric ratio of the two different compounds on thetheragnostic particle.

In one non-limiting embodiment, the engineered decoration proteinscomprise bacteriophage lambda gpD proteins, or functional mutantsthereof. The lambda gpD decoration protein is a monomer in solution butadds to the expanded viral particle as a trimer spike to each of the 140icosahedral three-fold axes (formed by the structural proteins) on theexpanded viral particle surface. In one embodiment, the engineered gpDproteins comprise a S42C substitution (SEQ ID NO: 10), which can be thesole decoration protein on the particle, or can be combined with wildtype gpD decoration proteins, or other gpD functional mutants, eachcomprising the one or more linked compounds (which may be the same ordifferent between the same or different gpD forms). For example,residues in the shell-bound gpD trimer spike that are exposed to solventinclude, but are not limited to Thr2, Ser3, Lys4, Glu5, Thr6, Phe7,Thr8, His9, Tyr10, Gln1, Pro12, Gln3, Gly14, Asn15, Ser16; Gly26, Gly27,Leu28, Ser29, Ala30, Lys31, Ala32; Asp41, Thr42, Ser43, Ser44, Arg45,Lys46; Asp51, Gly52, Thr53, Thr54, Asp55; Asp67, Gln68, Thr69, Ser70,Thr71, Thr72; Arg82, Tyr83, Glu84, Asp85; Glu90, Ala91, Ala92, Ser93,Asp94, Glu95, Thr96, Lys97, Lys98, Arg99, Thr100, and the N- andC-terminal residues of the protein. In one non-limiting embodiment, oneor more residue may be substituted with a Cys residue (including but notlimited to an S42C substitution), to facilitate binding of othercompounds to the decoration proteins. In another non-limitingembodiment, a non-natural amino acid, including but not limited toazidohomoalanine (Aha) can be incorporated using genetic manipulation ofthe gene to allow alternate means for chemical modification of thedecoration protein, including “click” chemistry.

The resulting theragnostic particles can be devoid of any packagedmaterial (“empty’) within the particle, or may include any desired cargomaterial packaged on the interior of the particle. For example, DMA isefficiently packaged into the decorated capsids and can modified tocarry specific genes of interest.

The theragnostic particles of the present invention can be used for anysuitable purpose. In one embodiment, the engineered decoration proteinsare linked to one or more therapeutic moieties (including but notlimited to small molecule drugs, therapeutic antibodies, glycoproteins,carbohydrate polymers, therapeutic nucleic acids (siRNA, antisense RNA,shRNA, gene therapy constructs, etc.), antigens (for use in vaccines),adjuvants (to stimulate an immune response), etc., and the particles canbe administered as a therapeutic to a subject in need of treatment. Inanother embodiment, synthetic polymers including PEG can be used toprovide “stealth” characteristics for immune evasion or pH sensitivepolymers for escape from cellular endosome compartments.

In various embodiments, the therapeutic is selected from the groupconsisting of alkylating agents, angiogenesis inhibitors, antibodies,antimetabolites, antimitotics, antiproliferatives, aurora kinaseinhibitors, apoptosis promoters (for example, Bcl-xL, Bcl-w and Bfl-1)inhibitors, activators of death receptor pathway, Bcr-Abl kinaseinhibitors, BiTE (Bi-Specific T cell Engager) antibodies, biologicresponse modifiers, cyclin-dependent kinase inhibitors, cell cycleinhibitors, cyclooxygenase-2 inhibitors, growth factor inhibitors, heatshock protein (HSP)-90 inhibitors, demethylating agents, histonedeacetylase (HDAC) inhibitors, hormonal therapies, immunologicals,inhibitors of apoptosis proteins (IAPs) intercalating antibiotics,kinase inhibitors, mammalian target of rapamycin inhibitors, microRNA'smitogen-activated extracellular signal-regulated kinase inhibitors,multivalent binding proteins, non-steroidal anti-inflammatory drugs(NSAIDs), poly ADP (adenosine diphosphate)-ribose polymerase (PARP)inhibitors, platinum chemotherapeutics, polo-like kinase (Plk)inhibitors, proteasome inhibitors, purine analogs, pyrimidine analogs,receptor tyrosine kinase inhibitors, retinoids/deltoids plant alkaloids,small inhibitory ribonucleic acids (siRNAs), topoisomerase inhibitorsand the like.

In another embodiment, the engineered decoration protein comprises oneor more diagnostic, localization, and/or imaging moieties (including butnot limited to fluorophores, radioactive tracers, dyes, diagnosticantibodies or other ligands, ligands for surface proteins on cells beingtargeted, etc.), and can be administered to a subject for diagnostic orimaging purposes. For example, the one or more compound may comprise adiagnostic or imaging agent. Many such imaging agents are known to thoseof skill in the art. Examples of imaging agents suitable for use in thedisclosed particles are radioactive isotopes, fluorescent molecules(including fluorescent proteins such as green fluorescent protein, redfluorescent protein, blue fluorescent protein, etc.), magnetic particles(including nanoparticles), metal particles (including nanoparticles),phosphorescent molecules, enzymes, antibodies, ligands, and combinationsthereof, while diagnostic agents may comprise a compound that is adiagnostic marker for a particular disorder bound to such an imagingagent. Methods for detecting and measuring signals generated by imagingagents are also known to those of skill in the art. For example,radioactive isotopes can be detected by scintillation counting or directvisualization; fluorescent molecules can be detected with fluorescentspectrophotometers: phosphorescent molecules can be detected with aspectrophotometer or directly visualized with a camera: enzymes can bedetected by detection or visualization of the product of a reactioncatalyzed by the enzyme; antibodies can be detected by detecting asecondary detection label coupled to the antibody.

In a further embodiment, the imaging agents can comprise a fluorescentimaging agent, while diagnostic agents may comprise a compound that is adiagnostic marker for a particular disorder bound to the fluorescentimaging agent. A fluorescent imaging agent is any chemical moiety thathas a detectable fluorescence signal. This imaging agent can be usedalone or in combination with other imaging agents. Examples of suitablefluorescent agents that can be used in the compositions and methodsdisclosed herein include, but are not limited to, quantum dots,fluorescein (FITC), 5-carboxyfluorescein-N-hydroxysuccinimide ester,5,6-carboxymethyl fluorescein, nitrobenz-2-oxa-1,3-diazol-4-yl (NBD),fluorescamine, OPA, NDA, indocyanine green dye, the cyanine dyes (e.g.,Cy3, Cy3.5, Cy5, Cy5.5 and Cy7),4-acetamido-4′-isothiocyanatostilbene-2,2′disulfonic acid, acridine,acridine isothiocyanate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonicacid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenylinaphthalimide-3,5disulfonate, N-(4-anilino-1-1-naphthyl)maleimide, anthranilamide,BODIPY, Brilliant Yellow, coumarin, 7-amino-4-methylcoumarin (AMC,Coumarin 120), 7-amino-4-trifluoromethylcoumarin (Coumaran 151),cyanosine, 4′,6-diaminidino-2-phenylindole (DAPI),5′,5″-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red),7-diethylamino-3-(4′-isothiocyanatophenyl)-4-methylcoumarindiethylenetriamine pentaacetate,4,4′-diisothiocyanatodihydro-stilbene-2,2′-disulfonic acid,4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid,5-[dimethylamino]naphthalene-1-sulfonyl chloride (DNS, dansylchloride),4-(4′-dimethylaminophenylazo)benzoic acid (DABCYL),4-dimethylaminophenylazophenyl-4′-isothiocyanatc (DABITC), cosin, cosinisothiocyanatc, crythrosin B, crythrosinc, isothiocyanate, ethidiumbromide, ethidium, 5-carboxyfluorescein (FAM),5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF),2′,7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE), fluoresceinisothiocyanate, IR144, IR1446, Malachite Green isothiocyanate,4-methylumbelliferone, ortho cresolphthalein, nitrotyrosine,pararosaniline, Phenol Red. B-phycoerythrin, o-phthaldialdehyde, pyrene,pyrene butyrate, succinimidyl 1-pyrene butyrate, Reactive Red 4(Cibacron[R] Brilliant Red 3B-A), 6-carboxy-X-rhodamine (ROX),6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloriderhodamine (Rhod), 5,6-tetramethyl rhodamine, rhodamine B, rhodamine 123,rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101,sulfonyl chloride derivative of sulforhodamine 101 (Texas Red).N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethyl rhodamine,tetramethyl rhodamine isothiocyanate (TRITC), riboflavin, rosolic acid,coumarin-6, and the like, including combinations thereof. Thesefluorescent imaging moieties can be obtained from a variety ofcommercial sources, including Molecular Probes, Eugene, Oreg. andResearch Organics, Cleveland, Ohio, or can be synthesized by those ofordinary skill in the art.

In another example, the imaging agents can comprise a Magnetic ResonanceImaging (MRI) agent, while diagnostic agents may comprise a compoundthat is a diagnostic marker for a particular disorder bound to the MRIagent. A MRI agent is any chemical moiety that has a detectable magneticresonance signal or that can influence (e.g., increase or shift) themagnetic resonance signal of another agent. This type of imaging agentcan be used alone or in combination with other imaging agent. Bycombining an MRI imaging agent and, for example, a fluorescent imagingagent, the resulting agent can be detected, imaged, and followed inreal-time via MRI. Other imaging agents include PET agents that can beprepared by incorporating an 18F or a chelator for 64Cu or 68Ga. Also,addition of a radionuclide can be used to facilitate SPECT imaging ordelivery of a radiation dose, while diagnostic agents may comprise acompound that is a diagnostic marker for a particular disorder bound tothe PET agent.

In some embodiments, the diagnostic agent is a diagnostic imaging agent,including but not limited to position emission tomography (PET) agents,computerized tomography (CT) agents, magnetic resonance imaging (MRI)agents, nuclear magnetic imaging agents (NMI), fluoroscopy agents andultrasound contrast agents. Such diagnostic agents include radioisotopesof such elements as iodine (I), including ¹²³I, ¹²⁵I, ¹³¹I etc., barium(Ba), gadolinium (Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P),including ³¹P, iron (Fe), manganese (Mn), thallium (Ti), chromium (Cr),including ⁵¹Cr, carbon (C), including ¹⁴C, or the like, fluorescentlylabeled compounds, or their complexes, chelates, adducts and conjugates.Any suitable PET agents can be used, including but not limited tocarbon-11, nitrogen-13, oxygen-15, fluorine-18,11C-metomidate, andglucose analogues thereof, including but not limited to fludeoxyglucose(a glucose analog labeled with fluorine-18.

The particles can also be used in viral research, such as to identifyprotein components required for viral assembly and mechanisticinterrogation of the assembly process.

In a further embodiment, the invention provides pharmaceuticalcompositions, comprising the theragnostic particles of any embodiment orcombination of embodiments of the invention and a pharmaceuticallyacceptable carrier. The pharmaceutically acceptable carrier isnon-toxic, biocompatible and is selected so as not to detrimentallyaffect the biological activity of the multimers (and any othertherapeutic agents combined therewith). Exemplary pharmaceuticallyacceptable carriers for peptides are described in U.S. Pat. No.5,211,657 to Yamada. The compositions may be formulated intopreparations in solid, semi-solid, gel, liquid or gaseous forms such astablets, capsules, powders, granules, ointments, solutions,suppositories, inhalants, and injections, allowing for oral, parenteral,or surgical administration. Suitable carriers for parenteral deliveryvia injectable, infusion, or irrigation and topical delivery includedistilled water, physiological phosphate-buffered saline, normal orlactated Ringer's solutions, dextrose solution. Hank's solution, orpropanediol. In addition, sterile, fixed oils may be employed as asolvent or suspending medium. For this purpose any biocompatible oil maybe employed including synthetic mono- or diglycerides. In addition,fatty acids, such as oleic acid, find use in the preparation ofinjectables. The carrier and agent may be compounded as a liquid,suspension, polymerizable or non-polymerizable gel, paste or salve. Thecarrier may also comprise a delivery vehicle to sustain (i.e., extend,delay, or regulate) the delivery of the agent(s) or to enhance thedelivery, uptake, stability, or pharmacokinetics of the therapeuticagent(s). Such a delivery vehicle may include, by way of non-limitingexample, microparticles, microspheres, nanospheres, or nanoparticlescomposed of proteins, liposomes, carbohydrates, synthetic organiccompounds, inorganic compounds, polymeric or copolymeric hydrogels, andpolymeric micelles. Suitable hydrogel and micelle delivery systemsinclude the PEO:PHB:PEO copolymers and copolymer/cyclodextrin complexesdisclosed in International Publication No. WO 2004/009664 A2, and thePEO and PEO/cyclodextrin complexes disclosed in U.S. Publication No.2002/0019369 A1. Such hydrogels may be injected locally at the site ofintended action, or subcutaneously or intramuscularly to form asustained release depot.

For intrathecal (IT) or intracerebroventricular (ICV) delivery,appropriately sterile delivery systems (e.g., liquids: gels,suspensions, etc.) can be used to administer the compositions. For oraladministration of non-peptidergic agents, the compositions may becarried in an inert filler or diluent such as sucrose, cornstarch, orcellulose.

The compositions of the present invention may also include biocompatibleexcipients, such as dispersing or wetting agents, suspending agents,diluents, buffers, penetration enhancers, emulsifiers, binders,thickeners, flavoring agents (for oral administration). Exemplaryformulations can be parenterally administered as injectable dosages of asolution or suspension of the multimer in a physiologically acceptablediluent with a pharmaceutical carrier that can be a sterile liquid suchas water, oils, saline, glycerol, or ethanol. Additionally, auxiliarysubstances such as wetting or emulsifying agents, surfactants, pHbuffering substances and the like can be present in compositionscomprising modified polypeptides. Additional components ofpharmaceutical compositions include petroleum (such as of animal,vegetable, or synthetic origin), for example, soybean oil and mineraloil. In general, glycols such as propylene glycol or polyethylene glycolare preferred liquid carriers for injectable solutions.

The pharmaceutical composition can also be administered in the form of adepot injection or implant preparation that can be formulated in such amanner as to permit a sustained or pulsatile release of the multimersand other therapeutic (if present).

The pharmaceutical composition may further comprise (a) a lyoprotectant;(b) a surfactant; (c) a bulking agent; (d) a tonicity adjusting agent;(e) a stabilizer, (f) a preservative and/or (g) a buffer. In someembodiments, the buffer in the pharmaceutical composition is a Trisbuffer, a histidine buffer, a phosphate buffer, a citrate buffer or anacetate buffer. The pharmaceutical composition may also include alyoprotectant, e.g. sucrose, sorbitol or trehalose. In certainembodiments, the pharmaceutical composition includes a preservative e.g.benzalkonium chloride, benzethonium, chlorohexidine, phenol, m-cresol,benzyl alcohol, methylparaben, propylparaben, chlorobutanol, o-cresol,p-cresol, chlorocresol, phenylmercuric nitrate, thimerosal, benzoicacid, and various mixtures thereof. In other embodiments, thepharmaceutical composition includes a bulking agent, like glycine. Inyet other embodiments, the pharmaceutical composition includes asurfactant e.g., polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, polysorbate-80 polysorbate-85, poloxamer-188, sorbitanmonolaurate, sorbitan monopalmitate, sorbitan monostearate, sorbitanmonooleate, sorbitan trilaurate, sorbitan tristearate, sorbitantrioleaste, or a combination thereof. The pharmaceutical composition mayalso include a tonicity adjusting agent, e.g., a compound that rendersthe formulation substantially isotonic or isoosmotic with human blood.Exemplary tonicity adjusting agents include sucrose, sorbitol, glycine,methionine, mannitol, dextrose, inositol, sodium chloride, arginine andarginine hydrochloride. In other embodiments, the pharmaceuticalcomposition additionally includes a stabilizer, e.g., a molecule which,when combined with a protein of interest substantially prevents orreduces chemical and/or physical instability of the protein of interestin lyophilized or liquid form. Exemplary stabilizers include sucrose,sorbitol, glycine, inositol, sodium chloride, methionine, arginine, andarginine hydrochloride. The pharmaceutical composition can be packagedin any suitable manner.

In a third aspect, the present invention provides isolated recombinantprotein comprising or consisting of the amino acid sequence of gpD(S42C)(SEQ ID NO: 10). As demonstrated in the examples that follow, gpD(S42C)is a particularly useful decoration protein. The crystal structure ofthe gpD trimer spike reveals that Ser42 is positioned at the apex of thespike in all three subunits (FIG. 8A). Modification of this residue thusplaces any linked compound projecting away from the capsid surface andinto solution for optimal display, and with minimal insult to gpD spikeassembly and shell integrity. There are no other cysteine residues inthe native protein, and thus the functional gpD mutant provides a uniquesite for chemical linkage via any suitable means, such as usingmaleimide-based tags. The use of these and other linking chemistries tolink two compounds to each other is well known in the art, and examplesare provided below.

In another embodiment, the invention provides isolated recombinantprotein comprising or consisting of the amino acid sequence of gpD asmodified by an amino acid substitution at one or more residues selectedfrom the group consisting of Thr2, Ser3, Lys4, Glu5, Thr6, Phe7, Thr8,His9, Tyr10, Gln11, Pro12, Gln13, Gly14, Asn15, Ser16; Gly26, Gly27,Leu28, Ser29, Ala30, Lys31, Ala32; Asp41, Thr42, Ser43, Ser44, Arg45,Lys46; Asp51, Gly52, Thr53, Thr54, Asp55; Asp67, Gln68, Thr69, Ser70,Thr71, Thr72; Arg82, Tyr83, Glu84, Asp85; Glu90, Ala91, Ala92, Ser93,Asp94, Glu95, Thr96, Lys97, Lys98, Arg99, and Thr100. In one embodiment,the one or more wild type amino acids are substituted with a Cys residueand/or an azidohomoalanine (Aha) residue.

In a further embodiment, a composition is provided comprising (a)isolated recombinant protein comprising or consisting of the amino acidsequence of SEQ ID NO: 10 (gpD(842C)); and (b) one or more compoundslinked to the recombinant protein via the cysteine at position 42. Thecompositions according to this embodiment are particularly useful forconstructing theragnostic particles according to the present invention.The compound may be any suitable compound, including but not limited tocompounds selected from the group consisting of nucleic acids, lipids,carbohydrates, polypeptides, polymers, organic molecules, or inorganicmolecules, or combinations thereof. In a further embodiment, thecompound is a non-proteinaceous compound, such as nucleic acids, lipids,carbohydrates, polymers, organic molecules, inorganic molecules (e.g.magnetic beads and quantum dots, among others), or combinations thereof.In a further embodiment, the composition comprises a plurality of suchrecombinant proteins with linked compounds, wherein the linked compoundsmay be the same or different from one recombinant protein to the next inthe plurality.

In a further aspect, the invention provides isolated nucleic acidsencoding the recombinant gpD(S42C) protein of the invention. The nucleicacids may comprise RNA or DNA, and can be prepared and isolated usingstandard molecular biological techniques, based on the teachings herein.The nucleic acids may comprise additional domains useful for promotingexpression and/or purification of the encoded protein, including but notlimited to polyA sequences, modified Kozak sequences, and sequencesencoding epitope tags, export signals, and secretory signals, nuclearlocalization signals, and plasma membrane localization signals.

In a further aspect, the present invention provides recombinantexpression vectors comprising the nucleic acid encoding the recombinantgpD(S42C) protein operatively linked to a promoter. “Recombinantexpression vector” includes vectors that operatively link a nucleic acidcoding region or gene to any promoter capable of effecting expression ofthe gene product. The promoter sequence used to drive expression of thedisclosed nucleic acids in a mammalian system may be constitutive(driven by any of a variety of promoters, including but not limited to,CMV, SV40. RSV, actin, EF) or inducible (driven by any of a number ofinducible promoters including, but not limited to, tetracycline,ecdysone, steroid-responsive). The construction of expression vectorsfor use in transfecting prokaryotic cells is also well known in the art,and thus can be accomplished via standard techniques. (See, for example,Sambrook, Fritsch, and Maniatis, in: Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Laboratory Press, 1989; Gene Transfer andExpression Protocols, pp. 109-128, ed. E. J. Murray, The Humana PressInc., Clifton, N.J.), and the Ambion 1998 Catalog (Ambion, Austin,Tex.). The expression vector must be replicable in the host organismseither as an episome or by integration into host chromosomal DNA, andmay comprise any other components as deemed appropriate for a given use,including but not limited to selection markers such as anantibiotic-resistance gene.

In a still further aspect, the present invention provides host cellscomprising the recombinant expression vectors disclosed herein, andprogeny thereof, wherein the host cells can be either prokaryotic oreukaryotic. The cells can be transiently or stably transfected. Suchtransfection of expression vectors into prokaryotic and eukaryotic cellscan be accomplished via any technique known in the art, including butnot limited to standard bacterial transformations, calcium phosphateco-precipitation, electroporation, or liposome mediated-, DEAE dextranmediated-, polycationic mediated-, or viral mediated transfection. (See,for example, Molecular Cloning: A Laboratory Manual (Sambrook, et al.,1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: AManual of Basic Technique, 2^(nd)Ed. (R. I. Freshney, 1987. Liss, Inc.New York, N.Y.). Techniques utilizing cultured cells transfected withexpression vectors to produce quantities of polypeptides are well knownin the art.

The description of embodiments of the disclosure is not intended to beexhaustive or to limit the disclosure to the precise form disclosed.While specific embodiments of, and examples for, the disclosure aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize.

All of the references cited herein are incorporated by reference.Aspects of the disclosure can be modified, if necessary, to employ thesystems, functions and concepts of the above references and applicationto provide yet further embodiments of the disclosure. These and otherchanges can be made to the disclosure in light of the detaileddescription.

Specific elements of any of the foregoing embodiments can be combined orsubstituted for elements in other embodiments. Furthermore, whileadvantages associated with certain embodiments of the disclosure havebeen described in the context of these embodiments, other embodimentsmay also exhibit such advantages, and not all embodiments neednecessarily exhibit such advantages to fall within the scope of thedisclosure. Accordingly, the disclosure is not limited.

EXAMPLE 1

The assembly of “complex” DNA viruses such as the herpesviruses and manytailed bacteriophages includes a DNA packaging step where the viralgenome is inserted into a pre-formed procapsid shell. Packaging triggersa remarkable capsid expansion transition that results in thinning of theshell and an increase in capsid volume to accept the full-length genome.This transition is considered irreversible; however, here we demonstratethat the phage λ procapsid can be expanded with urea in vitro and thatthe transition is fully reversible. This provides an unprecedentedopportunity to evaluate the thermodynamic features of this fascinatingand essential step in virus assembly. We show that urea-triggeredexpansion is highly cooperative and strongly temperature dependent.Thermodynamic analysis indicates that the free energy of expansion isinfluenced by magnesium concentration (3-13 kcal/mol in the presence of0.2-10 mM Mg²⁺) and that significant hydrophobic surface area is exposedin the expanded shell. Conversely, Mg²⁺ drives the expanded shell backto the procapsid conformation in a highly cooperative transition that isalso temperature dependent and strongly influenced by urea. Wedemonstrate that the gpD decoration protein adds to the urea-expandedcapsid, presumably at hydrophobic patches exposed at the three-fold axesof the expanded capsid lattice. The decorated capsid is biologicallyactive and sponsors packaging of the viral genome in vitro. The roles ofdivalent metal and hydrophobic interactions in controllingpackaging-triggered expansion of the procapsid shell are discussed inrelation to a general mechanism for DNA-triggered procapsid expansion inthe complex dsDNA viruses.

Introduction

The pathways for the assembly of an infectious virus from macromolecularprecursors are remarkably similar in all of the complex double strandedDNA (dsDNA) viruses both eukaryotic and prokaryotic^(1;2). Inparticular, the DNA replication, procapsid assembly, and genomepackaging pathways are strongly conserved in the herpesvirus groups andin many bacteriophages^(3;4;5;6;7). In these cases, a terminase enzymespecifically recognizes viral DNA and the terminase motor translocatesthe duplex into the interior of a pre-formed procapsid^(3;4;5;6). DNApackaging triggers a major reorganization of the proteins assembled intothe procapsid shell, which typically results in expansion of the shellinto a thinner, more angularized icocahedral structure^(8;9;10).Bacteriophage lambda (λ) has been extensively characterized genetically,biochemically, and structurally and provides an ideal system in which todefine the molecular details of genome packaging^(7;11;12).

Assembly of the λ procapsid follows an ordered pathway that is generallyconserved from phage to the herpesviruses. Briefly, assembly initiateswith self-association of the portal protein (gpB) into a dodecamericring structure^(12;13;14;15). This nucleates polymerization of the majorcapsid protein (gpE) into an icosahedral shell, chaperoned byco-polymerization with the scaffolding protein (gpNu3)^(16;17;18;19). Alimited number of viral protease proteins (gpC) are also incorporatedinto the nascent procapsid interior, which auto digests, degrades thescaffold protein, and removes 20 residues from the N-terminus of roughlyhalf of the portal proteins^(13;14). The proteolysis products exit thestructure to afford the mature procapsid composed of a portal ringsituated at a unique vertex of the icosahedral shell; this portal vertexprovides a hole through which viral DNA can enter during packaging andexit during infection.

Genome packaging represents the intersection of the DNA replication andprocapsid assembly pathways^(3;7). The terminase enzyme specificallyrecognizes viral DNA and then binds to the portal vertex of an emptyprocapsid (FIG. 1). This activates the terminase motor, whichtranslocates DNA into the procapsid interior, fueled by ATP hydrolysis.Upon packaging ˜15 kb DNA the procapsid undergoes an expansion process,which involves a significant reorganization of the capsid proteinsassembled into the shell (FIG. 1; discussed furtherbelow)^(7;11;20;21;22;23). Conventional wisdom dictates that procapsidexpansion represents “a major irreversible change in the assembledcapsid proteins of the procapsid shell”^(22;24;25).

In a number of viral systems, this irreversible transition can beartificially triggered in vitro using temperature, pH, ordenaturants^(26;27;28;29;30;31). In this study we examine urea-triggeredexpansion of the λ procapsid and report the surprising observation thatthe transition is fully reversible. The equilibrium is strongly affectedby urea concentration. magnesium concentration, salt, and temperature.We present physical, biochemical, and structural studies thatcharacterize this transition and confirm that the urea-expandedstructures faithfully recapitulate those generated by DNA packaging invivo. The relevance of these studies with respect to a general mechanismfor DNA-triggered procapsid expansion in the complex dsDNA viruses isdiscussed.

Results

Urea Triggers Expansion of the Lambda Procapsid.

Expansion of the λ procapsid in vivo is triggered upon packaging of ˜15kb duplex DNA (FIG. 1)^(20;21;23). We examined a variety of approachesto artificially expand procapsids to study this transition in vitro. Incontrast to other viral systems, neither pH in the range of 3-9 nor heatin the range of 4° C. to 50° C. are effective in promoting expansion.Prior work has demonstrated that the λ procapsid can be artificiallyexpanded by incubation with four molar urea for 30 minutes on ice²⁸.Here we recapitulate this result and demonstrate that incubation of ourpurified procapsids in 2.5 molar urea on ice for 15 minutes triggersprocapsid expansion (FIG. 2A). Due to the time required for analysis byagarose gel, an accurate quantitation of the reaction rate is notpossible using this assay. We note, however, that procapsid expansion isrelatively rapid and essentially complete in ˜1 minute by gel assay(data not shown). Interestingly, urea-triggered procapsid expansion isstrongly temperature dependent. While procapsids expand rapidly andcompletely on ice, the transition is strongly inhibited by elevatedtemperature (FIG. 2B). We considered that this might reflect a kineticeffect; however, urea-triggered expansion was not observed even after 24hours at 25° C. Urea-triggered expansion is also inhibited by NaCl in aconcentration dependent manner (FIG. 2C). Salt inhibition is likelyresponsible for the observation that the λ procapsid does not expand inthe presence of 4 molar guanidinium hydrochloride. For the remainder ofthis work we will use the term “procapsid” to describe the contractedshell (●) and the term “capsid” to describe the expanded, angularizedstructure (

; sec FIG. 2A).

Expansion of the Lambda Procapsid is Reversible.

Previous studies reported that dialysis of urea from the reactionmixture affords a preparation of λ capsids that remain in the expandedstate²⁸. In contrast, we observe that the structures contract back tothe procapsid state when urea is removed by buffer exchange into TMBbuffer (FIG. 2A). Close inspection of the published data reveals thatthe primary difference between our studies and previous work is thebuffer used to remove urea from the sample. This was investigated and weshow that the capsids remain in the expanded state when exchanged intothe TMSO buffer used in the prior studies (10 mM Tris buffer, pH 8,containing 1 mM MgSO₄ and 10 mM NaN₃). In contrast, the expanded shellscontract back to the procapsid conformation when exchanged into ourstandard TMB buffer (50 mM Tris buffer, pH 8, containing 15 mM MgCl₂ and7 mM β-ME) (FIG. 2A). It is generally accepted that procapsid expansionis an irreversible process in virus development and this surprisingobservation was more fully explored.

Each of the components in the two buffers was individually examined,which reveals that the increased concentration of Mg²⁺ in our TMB bufferis responsible for driving the expanded structure back to the procapsidstate; none of the other components has any effect. We next evaluatedother metals and the data presented in Table 1 demonstrate that capsidcontraction is driven by all of the divalent metals examined. Incontrast, none of the monovalent salts have any effect at similarconcentrations. Finally, we examined the extent to which the transitionis reversible. Procapsids that had been previously expanded with ureaand then contracted by buffer exchange into TMB buffer (15 mM Mg²⁺) wereagain expanded in urea and again buffer exchanged into TMB buffer. Thedata presented in FIG. 2D demonstrate that the λ procapsid can berepeatedly expanded and contracted in solution by urea and Mg²⁺,respectively.

TABLE 1 Di-Valent Metals Drive Contraction to the ProcapsidConformation. Procapsids were expanded in 2.5 M urea on ice for 15minutes and then exchanged into buffer containing 1 mM MgCl₂ and theindicated salt at 15 mM, unless otherwise indicated. In all cases, thepreparations contained either exclusively expanded capsid or contractedprocapsid structures, as indicated. Salt (15 mM) Capsid State MgCl₂Contracted MgSO₄ Contracted CaCl₂ Contracted BaCl₂ Contracted MnCl₂Contracted Ni(NO₃)₂ Contracted ZnCl₂ Contracted NaCl Expanded Naphosphate Expanded KCl Expanded K glutamate Expanded 100 mM NaClExpanded 500 mM NaCl Contracted

Thermodynamic Analysis of Urea-Triggered Procapsid Expansion.

The data presented above demonstrate that urea-triggered expansion ofthe λ procapsid is reversible. Moreover, close inspection of the gelsfails to reveal any evidence of intermediate states between thecontracted procapsid and expanded capsid conformations (see FIG. 2).These observations suggest that the expansion reaction may be modeled asa reversible, two-state transition and we adapted analytical toolsdeveloped to characterize two-state protein unfolding reactions³².Procapsids were expanded as described in Materials and Methods exceptthat the concentration of MgCl₂ and urea was varied as indicated. In thepresence of 0.2 mM MgCl₂, procapsid expansion is urea-concentrationdependent and strongly cooperative (FIG. 3A). Analysis of the dataaccording to a reversible, two-state transition (Equation 1)³² affords afree energy of expansion, ΔG(H₂O)˜3 kcal/mol. We note that the expansiontransition under these conditions is extremely facile and occurs withessentially no pre-transition baseline. The error associated with thisanalysis is thus rather large; ΔG(H₂O)=3.3±4.4 kcal/mol).

As described above, Mg²⁺ drives the expanded shell back to thecontracted procapsid conformation. We interpreted the data to indicatethat urea and Mg²⁺ have antagonistic effects on the conformation of theλ capsid and we directly tested this hypothesis. The aboveurea-expansion study was repeated except that MgCl₂ was included in thereaction mixture at 3 mM or 10 mM. The data clearly indicate that Mg²⁺antagonizes urea-triggered procapsid expansion in aconcentration-dependent manner (FIG. 3A) and that the concentration ofurea required to expand half of the procapsids ([urea]_(1/2)) isstrongly affected by Mg²⁺ (FIG. 3B). Analysis of the expansion dataaccording to Equation 1 affords a ΔG(H₂O) of 10.0±3.0 kcal/mol and13.4±2.4 kcal/mol in the presence of 3 mM and 10 mM Mg²⁺, respectively(FIG. 3C). For comparison, “typical” protein unfolding reactions report[urea]_(1/2) between 3-6 molar and a ΔG(H₂O) that ranges between 5-15kcal/mol^(32;33;34;35). Finally, we note that the denaturant “m” valuefor the transition is relatively large and insensitive to theconcentration of Mg²⁺ in the reaction mixture (FIG. 3C). This isdiscussed further below.

Mg²⁺ Driven Capsid Contraction.

To further explore the antagonist effects of urea and Mg²⁺, apreparation of expanded capsids in buffer lacking both urea and Mg²⁺ wasprepared by buffer exchange. The expanded capsids were then incubated inthe presence of increasing concentrations of MgCl₂ and the fraction ofstructures that had contracted back to the procapsid state wasquantified by gel assay. The data presented in FIG. 4 demonstrate thatMg²⁺ drives the expanded shell back to the procapsid conformation in aconcentration dependent and strongly cooperative manner; analysis of thedata according to Equation 3 affords a [Mg²⁺]_(1/2)=1.1±0.1 mM. We nextrepeated the experiment as described above except that 2.5 molar ureawas included in the reaction buffer. While Mg²⁺ can still drive thecontraction transition, significantly higher concentrations of divalentmetal are required; [Mg²⁺]_(1/2)=6.5±0.4 mM (FIG. 4).

Biological Activity of the Expanded λ Capsids.

We have demonstrated that the 1 procapsid can be reversibly expanded andcontracted in vitro, which has allowed a rigorous thermodynamiccharacterization of the transition (vide supra). Evaluation of thesedata with specific reference to virus assembly requires thaturea-triggered expansion mimic the natural pathway that is triggered byDNA packaging in vivo. The three most essential functions required ofprocapsids during DNA packaging are (i) the ability to bind theterminase motor and sponsor DNA packaging, (ii) the ability to bind thegpD decoration protein to the expanded capsid lattice, and (iii) thecapacity of the decorated shell to physically withstand the internalforces generated by the packaged λ genome.

Our lab has developed an in vitro DNA packaging assay where viral DNA ispackaged into purified procapsids in a defined biochemical assaysystem^(36;37). These studies have demonstrated that the packagingreaction is magnesium-dependent and that the optimal MgCl₂ concentrationis ˜5 mM. This presents a problem when trying to package into theexpanded capsids since they contract in the presence of >1 mM Mg²⁺ (FIG.4). Therefore, as a first step towards demonstrating the biologicalactivity of the urea-expanded capsids, we examined gpD binding to thecapsid shell. An expanded capsid preparation in 1 mM Mg²⁺ (no urea) wasprepared and purified gpD was added to the mixture. FIG. 5A clearlyshows that the gpD decoration protein efficiently adds to the expandedcapsid lattice. Several features of this interaction are noteworthy.First, while gpD binds to the expanded capsid. no interaction isobserved with unexpanded procapsid shell (FIG. 5A); this feature issimilarly observed in vivo^(7;38). Second, the gpD-decorated shellmigrates faster in the gel than does the expanded capsid alone. Thisindicates that gpD addition affords a more negatively charged capsidshell. Third. gpD stabilizes the expanded shell and the decoratedcapsids no longer contract in the presence of elevated Mg²⁺concentrations (Medina, Kruse, and Catalano, unpublished³⁹). Finally,electron micrographs demonstrate that the gpD decorated structures showthinning of the capsid shell and increased angularization of theicosahedral structure (FIG. 5B), as observed in vivo²³. We note that theexpanded shells are fragile and easily damaged during preparation for EManalysis (see FIG. 5B). In contrast. the gpD-decorated shells are robustand intact structures are evident in the micrographs.

To further demonstrate biological activity, we examined the genomepackaging activity of the gpD-decorated capsids. Terminase-mediatedpackaging of the λ genome into a capsid renders the duplex resistant toDNase and the packaging products are visualized on an agarose gel³⁶. Ourstandard DNA packaging assay was modified by pre-incubating the expandedcapsids with gpD and then adding the other reaction components requiredfor genome packaging. The data presented in FIG. 5C demonstrate that theexpanded. gpD coated capsids are fully competent for DNA packaging.Importantly, the only packaging product is the full-length λ genome(inset). This indicates that packaging into the urea-expanded capsids ishighly processive and that DNA-filled particles can withstand thetremendous internal forces generated by the tightly packaged, highlypressurized DNA genome^(20;40;41). These features mirror theobservations with bone fide λ capsids both in vitro^(20;40) and invivo^(7;14;38).

Discussion

The packaging of a viral genome into a pre-formed procapsid structure isan essential step in the assembly of complex dsDNA viruses. A universalfeature is that DNA packaging triggers maturation of the procapsid,often resulting in expansion and thinning of the shell to afford alarger, more angularized structure. Dogma contends that this step isirreversible so that unidirectional assembly of the viral particle isensured. This presumption is consistent with a number of in vitrostudies where procapsid expansion has been artificially triggered byusing pH, heat, or denaturants^(26;27;28;30;42;43;44); however, therehas been some indication that strict irreversibility may not necessarilybe the case. For instance, pH-induced expansion of the HK97 procapsidproceeds through a number of intermediates and there is evidence that atleast one transition may be partially (10%) reversible 45. That said,the fully expanded capsid structure is stable and does not contract backto the procapsid conformation. Thus, our demonstration thaturea-triggered expansion of the λ procapsid is fully reversible and thatthe expansion-contraction transition can be repeated for multiple cyclesis unexpected and quite remarkable.

Energetic Features of Procapsid Expansion.

We have demonstrated that Mg²⁺ and urea stabilize the contracted and theexpanded conformations of the λ capsid, respectively. We have furthershown that inter-conversion between the two structures is highlycooperative and fully reversible. These features allow a rigorousthermodynamic characterization of the expansion reaction, which has notbeen possible in any other system. Our data indicate that the freeenergy of urea-triggered procapsid expansion, ΔG(H₂O), is ˜13 kcal/molin the presence of 10 mM MgCl₂. This translates directly to ˜90picoNewtons*nanometer (pN·nm) work done to expand an individual capsid.It is generally presumed that packaging DNA into the procapsid interiorgenerates pressure that ultimately provides the energy, at least inpart, required to trigger expansion^(6;7;20). Previous single-moleculestudies suggest that expansion is triggered when the terminase motorinserts ˜15 kb duplex DNA into the procapsid (a magnesium concentrationof 10 mM was used in the laser tweezer studies) (see FIG. 1)²⁰. Themotor generates ˜5 pN force at this point^(46;47), which reflects theenergy required to condense the DNA into the confines of the procapsidinterior. To the first approximation, this force will generate apressure of 0.06 N/cm² on the inside of the shell. Expansion increasesthe diameter of the particle from 50 nm to 60 nm^(23;41), which resultsin a volume increase of 4.5×10²⁰ liters. This means that the mechanicalwork performed by the motor at the point of procapsid expansion is ˜70pN·nm per capsid (work=PΔV), which to the first approximationcorresponds quite well to the work required to expand the structure withurea in vitro (˜90 pN·nm, above). While admittedly qualitative innature, these simple calculations indicate that the free energy ofprocapsid expansion by urea is commensurate with the work performed bythe motor at the point of expansion during DNA packaging. This isdiscussed further below.

Magnesium Stabilization of the Procapsid—Possible Biological Role?

Divalent metals play an essential role in the assembly of many viruses.For example, Ca²⁺ plays an important role in the assembly andstabilization of polyoma virus^(48;49;50) and herpes virus⁵¹ particles.Similarly, early studies demonstrated an important role for Mg² in theassembly and stability of an infectious λ virus^(52;53). Chelation ofMg²⁺ with EDTA destroys λ infectivity and it has been proposed that Mg²⁺serves as a counter-ion to maintain DNA in the condensed state withinthe nucleocapsid⁵⁴. Stabilization of the procapsid conformation bydivalent metals as demonstrated here indicates that there is ametal-capsid interaction in addition to the metal-DNA interaction.Indeed, recent structural studies have identified putative metal-bindingsites localized at the 3-fold axes on the interior surface of the HK97procapsid⁵⁵. We suggest that this feature is recapitulated in λ and thatthis metal-capsid interaction is responsible. at least in part. forstabilizing the procapsid conformation. This model has some interestingpredictions.

In addition to the pressure generated by the packaged DNA (vide supra),it is presumed that duplex interactions with the inner surface of theprocapsid shell trigger the expansion transition; however, mechanisticdetails of these putative interactions remain obscure. We suggest thefollowing. Condensation of DNA within the capsid interior isenergetically unfavorable due, in part, to significant charge-repulsionby the closely packed phosphodiester backbone. Charge neutralization bypolyamines and divalent metals is required to ensure efficientcondensation and packaging of the genome^(54;56;57). The terminase motortranslocates over 600 base pairs/second into the capsidinterior^(20;58). and it is tempting to speculate that the rapidlypackaged duplex effectively strips the Mg²⁺ bound at the interior capsidsurface. This would serve to decrease the free energy requirement forexpansion of the shell, perhaps as much as 10 kcal/mol (FIG. 3), and topromote the transition. As shown here, expansion is accompanied byaddition of gpD to the capsid surface, which stabilizes the expandedshell and prevents contraction back to the procapsid state. In thissituation, divalent metals are free to bind to and stabilize thecondensed DNA. Work currently underway in our lab seeks to directlymeasure the duplex length requirement for procapsid expansion in thepresence of various divalent metals to directly test this hypothesis.

Procapsid Expansion Exposes Hydrophobic Surface Area.

We propose that the procapsid conformation is stabilized not only bydivalent metal, but also by hydrophobic interactions between the capsidproteins in the contacted shell (see FIG. 6). The expansion transitionrequires disruption of these interactions and exposure of hydrophobicpatches on the expanded capsid surface. This hypothesis is based in parton the observation that urea triggers procapsid expansion; while themechanism remains controversial, it is generally accepted that ureadisrupts hydrophobic interactions and effectively “solvates” hydrophobicresidues in water^(34;59;60). Thus, in analogy to protein unfolding,urea stabilizes the expanded capsid conformation and shifts theequilibrium towards the expanded state. The model is also consistentwith the observation that procapsid expansion is strongly inhibited bysalt and increased temperature, both of which increase the strength ofhydrophobic interactions. For instance, expansion occurs efficiently at4° C. but not at 25° C., a temperature range in which the strength ofhydrophobic interactions increase to a maximum at ˜20° C.^(59;61). Insum, our data are consistent with a transition that requires overcominghydrophobic interactions.

The model proposed in FIG. 6 is further consistent with the largedenaturant “m” value obtained in our thermodynamic analysis ofurea-triggered expansion. This value reflects the dependence of ΔG ondenaturant concentration, which is related to the heat capacity change(ΔCp). This in turn is related to the change in hydrophobic surfacearea; the larger the m, the greater hydrophobic surface area is exposedin the transition³³. The m value obtained here (>4kcal/mol·molar^(32;34;35;62)) is relatively large compared to mostprotein unfolding reactions (typically 1-3kcal/mol·molar^(32;34;35;62)). This indicates that a large hydrophobicsurface area is exposed upon capsid expansion. Importantly, while theΔG(H₂O) for expansion increases with increasing magnesium concentration,the denaturant m value remains relatively constant. This indicates thatwhile magnesium stabilizes the procapsid state, it does not affect thechange in hydrophobic surface area exposed upon transition to theexpanded conformation. In other words, the conformation of the expandedstate is the same regardless of the Mg²⁺ concentration, as would beexpected of a simple two-state transition.

Mechanism for Addition of gpD to the Expanded Capsid Lattice.

The terminase motor packages DNA to liquid crystalline density withinthe capsid, which generates over 20 atmospheres of internalpressure^(20;63;64;65). Viral decoration proteins add to the surface ofthe expanded capsid structure to stabilize the shell against thetremendous internal forces generated by the tightly packagedDNA^(20;40;41). Specifically, the λ gpD protein assembles as a trimericspike at the three-fold axes of the expanded capsid lattice^(23;41).While gpD is dispensable for packaging of duplexes up to ˜40 kb.packaging of larger duplexes requires gpD to maintain capsidintegrity⁴⁰.

Structural studies indicate that the base of the gpD trimer ishydrophobic and it has been proposed that the spike interacts withhydrophobic patches on the capsid surface^(41;66;67); however,biochemical evidence for this model is lacking. Our data indicate thatprocapsid expansion is associated with exposure of hydrophobic patcheson the capsid surface. We propose that these patches, which are buriedin the procapsid conformation, are exposed at the icosahedral three-foldaxes of the expanded shell and provide a nucleation site for assembly ofthe gpD trimer. Importantly, gpD adds to the surface of the expandedcapsid efficiently at room temperature but only poorly at 4° C. (datanot shown; see³⁹); this temperature dependence is consistent with anincrease in hydrophobic binding energy within this temperature range(vide supra)^(59;61).

Although our data show that expansion of the λ procapsid is fullyreversible, gpD addition effectively locks the capsid shell into theexpanded state. Indeed, large-scale conformational changes accompany DNApackaging in all of the complex dsDNA viruses and this is intrinsicallyirreversible. In vivo. these transitions are further made irreversibleby proteolytic cleavage events, capsid protein cross-linking events, orthe addition of decoration proteins as is observed in λ. Subsequently,“finishing” proteins add in an ordered, step-wise sequence, interactionsthat are nucleated by addition of the previous protein. In this manner,the particle progresses to the next step of assembly to minimize“off-pathway” intermediates and ensures fidelity in the assemblyprocess.

Why Do Procapsids Expand?

Procapsid expansion is a common feature in the packaging of viral DNAbut it is not clear why this is necessary. One possibility is that theassembly of a procapsid shell provides a mechanism for the terminasepackaging motor to select only packaging competent shells. This presumesthat pre-expanded capsids represent defective, off pathway intermediatesthat may result from aborted packaging events; however, our datademonstrate that the expanded. capsid is catalytically competent andpackages DNA quite efficiently. Indeed, this has similarly beendemonstrated in the bacteriophage T4 system^(68;69). Thus, while DNApackaging triggers the transition in vivo, expansion per se does notinfluence the capacity of the shell to accept the viral genome.

This begs the question as to why viruses utilize a procapsid structureat all. One possibility proposed by Johnson and co-workers is that theextensive interactions between the protein subunits assembled into themature capsid shell are unlikely to form in a single assembly step⁵⁵.This model suggests that the procapsid represents a metastableintermediate that is required for fidelity in shell assembly and thatexpansion is a consequence of the transition to the stable mature shell.Such a transition implies irreversibility; however, our data show thatthis need not be the case.

Conclusions.

We have demonstrated that magnesium and urea stabilize the contractedand expanded conformations of the λ capsid shell, respectively. The λsystem is unique in that expansion is fully reversible, which hasallowed thermodynamic characterization of the transition. Expansion ofthe shell is associated with exposed hydrophobic surface area to whichthe gpD decoration protein adds. This stabilizes the expanded capsidlattice, abrogates contraction, and provides structural integrity sothat the genome can be tightly packaged into the capsid interior. Thiswork further provides mechanistic insight into how DNA packagingtriggers shell expansion that is generalizable to the complex dsDNAviruses, both prokaryotic and eukaryotic.

Materials and Methods for Example 1

Materials and Methods.

Tryptone, yeast extract, and agar were purchased from DIFCO. Molecularbiology enzymes and mature λ DNA were purchased from New EnglandBiolabs. Chromatography media was purchased from GE Healthcare and ureawas purchased from Fisher Scientific. All other materials were of thehighest quality available. Unless otherwise stated, Tris buffers wereadjusted to the indicated pH at a temperature of 4° C. Bacterialcultures were grown in shaker flasks utilizing an Innova 4430incubator-shaker. All protein purifications utilized the AmershamBiosciences ÄKTApurifier™ core 10 System from GE Healthcare. UV-VISabsorbance spectra were recorded on a Hewlett-Packard HP8452Aspectrophotometer. Sucrose density gradients were prepared using aBiocomp Model 107 Gradient Master® and gradient centrifugation utilizeda Beckman L-90K ultracentrifuge with a SW 28 rotor.

Protein Purification.

λ terminase and the Escherichia coli Integration Host Factor (IHF) werepurified as previously described⁷⁰ ⁷¹. The gpD decoration protein waspurified by our published protocol³⁶, with modification. Briefly, gpDwas expressed from the pT7Cap vector and ammonium sulfate was added tothe cell lysis supernatant to 50%0/saturation. The mixture was gentlystirred on ice for 50 minutes and insoluble protein was removed bycentrifugation (9 K×g, 20 minutes). The supernatant was adjusted to 90%ammonium sulfate, stirred on ice for 50 minutes and insoluble proteinwas harvested by centrifugation (30 K×g, 30 minutes). The pellet wasresuspended in 20 mM Tris buffer, pH 8, containing 1 mM EDTA and 15 mMNaCl and the sample was applied to a DEAE column equilibrated with thesame buffer. The column flow-through fraction, which contained gpD, wasapplied to a S-300 gel filtration column equilibrated and developed with20 mM Tris buffer, pH 8, containing 1 mM EDTA, and 100 mM NaCl. The gpDcontaining fractions were pooled, dialyzed into 20 mM Tris buffer, pH 8,containing 1 mM EDTA and concentrated to ˜500 μM using an Amiconcentrifugal filter unit. All of our purified protein preparations werehomogenous as determined by SDS-PAGE (not shown).

Procapsid Purification.

λ procapsids were purified as described previously³⁶, but the purifiedpreparations contained variable amounts of pre-expanded procapsids. Toobtain a homogenous preparation of unexpanded procapsids, the sample wasfractionated on a 0.8% agarose gel, which readily separates expandedcapsids from unexpanded procapsids (see FIG. 2). The lower band wasexcised with a sterile razor blade and the unexpanded procapsids wereeluted from the gel slice into Buffer A (25 mM Tris base, 192 mMglycine, and 1 mM MgCl₂) using a Bio-Rad Model 422 Electro-eluter (100 Vfor 1 hour). The sample was dialyzed into TMB buffer (50 mM Tris buffer,pH 8, containing 15 mM MgCl₂ and 7 mM β-ME) and concentrated using anAmicon® centrifugal filter unit.

Procapsid Expansion and Buffer Exchange Protocol.

A freshly prepared stock solution of 8 molar urea in water was used forall of the expansion experiments. Unless otherwise specified, equalvolumes of purified procapsids and 8 molar urea were mixed to afford areaction mixture (20 μl) containing 30 nM procapsids in 10 mM Trisbuffer, pH 8. containing 2.5 molar urea and 3 mM MgCl₂. Subsequentmodification of the expansion reaction composition was accomplished by abuffer exchange protocol. Briefly, a sufficient volume of buffer wasadded to adjust the magnesium and urea concentrations as indicated ineach experiment and the mixture was then concentrated to its originalvolume using an Amicon® centrifugal filter unit. The expansion reactionmixtures were incubated on ice for 15 minutes and procapsid expansionanalyzed by agarose gel assay, as described below.

Analysis of Capsid Expansion and Contraction.

The samples were applied to a 0.8% agarose gel and electrophoresis wasperformed at 110 v for 100 minutes. The contracted procapsid (lower) andexpanded capsid (upper) bands were visualized by staining with Coomassiebrilliant blue. Video images of the de-stained gels were captured usingan EpiChemi³ darkroom system with a Hammamatsu camera (UVP BioimagingSystems) and the video images were quantified using either the LabWorks4.6 (UVP Bioimaging Systems) or the ImageQuant (Molecular Dynamics)software packages.

Thermodynamic Analysis of Urea-Triggered Procapsid Expansion.

Procapsid expansion in urea is a two-state, reversible transition and weadapted analytical tools developed to characterize reversible proteinunfolding transitions to analyze the data, as follows. The fraction ofprocapsids in the expanded state was quantified by agarose gel assay(above). The data were fit by nonlinear, least-squares analysis usingthe linear extrapolation method as outlined by Santoro and Bolen³²according to equation 1:

$\begin{matrix}{F_{E} = \frac{\begin{matrix}{\left( {{m_{P}*\lbrack U\rbrack} + b_{P}} \right) + {\left( {{m_{E}*\lbrack U\rbrack} + b_{E}} \right)*}} \\{\exp\left\lbrack {- \left( {\frac{\Delta\; G_{H_{2}O}}{RT} + \frac{m_{G}*\lbrack U\rbrack}{RT}} \right)} \right\rbrack}\end{matrix}}{1 + {\exp\left\lbrack {- \left( {\frac{\Delta\; G_{H_{2}O}}{RT} + \frac{m_{G}*\lbrack U\rbrack}{RT}} \right)} \right\rbrack}}} & (1)\end{matrix}$where F_(E) is the fraction of capsids in the expanded state as afunction of the urea concentration ([U]). The values m_(P) and m_(E)represent the slopes and b_(P) and b_(F) represent the y-intercepts ofthe pre- and post-transition baselines, respectively. R is the ideal gasconstant and T is the temperature (kelvin). The free energy of procapsidexpansion (ΔG_(H) ₂ _(O); kcal/mol) and the denaturant m value (m_(G);kcal/mol·molar) represent the intercept and slope, respectively, of thelinear dependence of the expansion transition energy as a function ofdenaturant concentration. Each data set was fit to equation 1 withm_(P), b_(P), m_(E), b_(E), ΔG_(H) ₂ _(O), and m_(G) as parameters usingthe IGOR® graphics/analysis package (WaveMetrics, Lake Oswego, Oreg.).The urea-expansion data were also fit according to equation 2:

$\begin{matrix}{F_{E} = {{Base} + \left\lbrack \frac{Max}{1 + {\exp\left( \frac{\lbrack{urea}\rbrack_{1/2} - \lbrack{urea}\rbrack}{m_{T}} \right)}} \right\rbrack}} & (2)\end{matrix}$where F_(E) is the fraction of capsids in the expanded state as afunction of the urea concentration ([urea]). Base and Max represent thepre- and post-transition baselines, m_(T) is the transition slope, and[urea]_(1/2) is the concentration of urea required to expand half of theprocapsids to the expanded state. Each data set was fit to equation 2with Base, Max. C_(1/2) and m_(T) as parameters using the IGOR®graphics/analysis package (WaveMetrics, Lake Oswego, Oreg.).

Analysis of Mg²⁺-Triggered Capsid Contraction.

Expanded capsids were prepared as described above in the absence andpresence of urea at the indicated concentration. The samples wereincubated in the presence of the indicated concentration of MgCl₂ atroom temperature for five minutes and the fraction of expanded capsidstructures quantified by agarose gel and video densitometry as describedabove. The data were fit by nonlinear, least-squares analysis accordingto equation 3:

$\begin{matrix}{F_{E} = {{Base} + \left\lbrack \frac{Max}{1 + {\exp\left( \frac{\lbrack{Mg}\rbrack_{1/2} - \lbrack{Mg}\rbrack}{m_{T}} \right)}} \right\rbrack}} & (3)\end{matrix}$where F_(E) is the fraction of capsids in the expanded state as afunction of the Mg²⁺ concentration, [Mg]. Base and Max represent thepre- and post-transition baselines, m_(T) is the transition slope, and[Mg]_(1/2) is the concentration of Mg²⁺ required to contract half of theexpanded shells to the procapsid state. Each data set was fit toequation 2 with Base, Max, [Mg]_(1/2) and m_(T) as parameters using theIGOR® graphics/analysis package (WaveMetrics, Lake Oswego, Oreg.).

DNA Packaging into Expanded Capsids.

The in vitro DNA packaging reaction was performed as describedpreviously³⁶ with modification. Briefly, purified procapsids (40 nMcapsid; 16.6 μM gpE capsid protein) were expanded as described above andthen buffer exchanged into 10 mM Tris buffer, pH 8. Purified gpD wasthen added to a final concentration of 15 μM and the mixture wasincubated for 30 minutes at room temperature. The gpD-coated, expandedcapsids were added to a reaction mixture containing 50 mM Tris buffer,pH 7.4, containing 9 mM NaCl, 5 mM MgCl₂, 2 mM spermidine, 1.3 mM β-ME,1 mM ATP, 100 nM IHF, and 2 nM mature λ DNA. The packaging reaction (20μl) was initiated with the addition of terminase holoenzyme to a finalconcentration of 100 nM and allowed to proceed for 30 minutes at roomtemperature. DNase (10 μg/ml) was then added to the reaction mixture andincubated at room temperature for five minutes. The DNase reaction wasstopped with the addition of phenol:chloroform (21 μl) and the aqueouslayer was removed and loaded onto a 0.8% agarose gel. Packaged (DNaseresistant) DNA was quantified by video densitometry as previouslydescribed³⁶.

REFERENCES FOR EXAMPLE 1

-   1. Calendar, R. & Abedon, S. T. (2006). The Bacteriophages, Oxford    University Press, New York, N.Y.-   2. Roizman, B. & Palese, P. (1996). Multiplication of Viruses: An    Overview. In Fields Virology Third edit. (Fields, B. N.,    Knipe, D. M. & Howley, P. M., eds.), pp. 101-11. Lippincott-Raven,    New York, N.Y.-   3. Rao, V. B. & Feiss, M. (2008). The bacteriophage DNA packaging    motor. Annu Rev Genet 42, 647-81.-   4. Roizman, B., Knipe, D. M. & Whitley, R. J. (2007). Herpes Simplex    Viruses. In Fields Virology Fifth edit. (Knipe, D. M. & Howley, P.    M., eds.), pp. 2501-2602. Lippincott. Williams and Wilkins,    Philadelphia, Pa.-   5. Catalano, C. E. (2005). Viral Genome Packaging Machines: An    Overview. In Viral Genome Packaging Machines: Genetics, Structure,    and Mechanism (Catalano. C. E., ed.). pp. 1-4. Kluwer    Academic/Plenum Publishers, New York, N.Y.-   6. Baines. J. D. & Weller, S. K. (2005). Cleavage and Packaging of    Herpes Simplex Virus 1 DNA. In Viral Genome Packaging Machines:    Genetics, Structure, and Mechanism (Catalano, C. E., ed.), pp.    135-149. Kluwer Academic/Plenum Publishers. New York, N.Y.-   7. Feiss, M. & Catalano, C. E. (2005). Bacteriophage Lambda    Terminase and the Mechanism of Viral DNA Packaging. In Viral Genome    Packaging Machines: Genetics. Structure, and Mechanism (Catalano, C.    E., ed.), pp. 5-39. Kluwer Academic/Plenum Publishers, New York,    N.Y.-   8. Jardine, P. J. & Anderson, D. L. (2006). DNA Packaging in    Double-Stranded DNA Phages. In The Bacteriophages 2nd edit.    (Calendar, R. & Abedon, S. T., eds.), pp. 49-65. Oxford University    Press, New York, N.Y.-   9. Baines, J. D. & Duffy, C. (2006). Nucloeocapsid Assembly and    Envelopment of Herpes Simplex Virus. In Alpha Herpesviruses:    Molecular and Cellular Biology (Sandri-Goldin, R. M., ed.), pp.    175-204. Caister Academic Press, Norfolk, Va.-   10. Alonso, J. C., Tavares, P., Lurz, R. & Trautner, T. A. (2006).    Bacteriophage SPPI. In The Bacteriophages 2nd edit. (Calendar, R. &    Abedon, S. T., eds.), pp. 331-349. Oxford University PRess, New    York, N.Y.-   11. Hendrix, R. W. & Casjens. S. (2006). Bacteriophage Lambda and    its Genetic Neighborhood. In The Bacteriophages 2nd edit.    (Calendar, R. & Abedon, S. T., eds.), pp. 409-447. Oxford University    Press, New York, N.Y.-   12. Hendrix, R. W., Roberts, J. W., Stahl, F. W. & Weisberg, R. A.    (1983). Lamba II, Cold Spring Harbor Laboratory, Cold Spring Harbor,    N.Y.-   13. Medina, E., Wieczorek, D. J., Medina, E. M., Yang, Q., Feiss, M.    & Catalano, C. E. (2010). Assembly and Maturation of the    Bacteriophage Lambda Procapsid: gpC Is the Viral Protease. J. Mol.    Biol. 401, 813-830.-   14. Georgopoulos, C., Tilly, K. & Casjens, S. (1983). Lambdoid Phage    Head Assembly. In Lambda II (Hendrix, R. W., Roberts, J. W.,    Stahl, F. W. & Weisberg, R. A., eds.), pp. 279-304. Cold Spring    Harbor Laboratory, Cold Spring Harbor, N.Y.-   15. Kochan, J. & Murialdo, H. (1983). Early intermediates in    Bacteriophage Lambda Prohead Assembly. II. Identification of    Biologically Active Intermediates. Virology 131, 100-115.-   16. Medina. E. M., Andrews, B. T., Nakatani, E. & Catalano, C. E.    (2011). The Bacteriophage Lambda gpNu3 Scaffolding Protein is an    Intrinsically Disordered and Biologically Functional Procapsid    Assembly Catalyst. Journal of Molecular Biology 412, 723-736.-   17. Johnson, J. E. (2010). Virus Particle Maturation: Insights into    Elegantly Programmed Nanomachines. Current Opinion in Structural    Biology 20, 210-216.-   18. Fane, B. A. & Prevelige, P. E. (2003). Mechanism of    Scaffolding-Assisted Viral Assembly. In Virus Structure (Wah, C. &    John, E. J., eds.). Vol. Volume 64. pp. 259-299. Academic Press,    Oxford, UK.-   19. Dokland, T. (1999). Scaffolding Proteins and their Role in Viral    Assembly. Cellular and Molecular Life Sciences 56, 580-603.-   20. Fuller, D. N., Raymer, D. M., Rickgauer, J. P., Robertson, R.    M., Catalano, C. E., Anderson, D. L., Grimes, S. & Smith, D. E.    (2007). Measurements of single DNA molecule packaging dynamics in    bacteriophage lambda reveal high forces, high motor processivity.    and capsid transformations. J Mol Biol 373, 113-22.-   21. Hohn, T., Wurtz, M. & Hohn, B. (1976). Capsid Transformation    During Packaging of Bacteriophage Lambda DNA. Phil. Trans. R. Soc.    Lond. 276, 51-61.-   22. Hohn, T., Morimasa, T. & Tsugita, A. (1976). The Capsid Protein    of Bacteriophage Lambda and of its Prehead. Journal of Molecular    Biology 105, 337-342.-   23. Dokland, T. & Murialdo, H. (1993). Structural Transitions During    Maturation of Bacteriophage Lambda Capsids. J. Mol. Biol. 233,    682-694.-   24. Steven, A. C., Heymann, J. B., Cheng, N., Trus. B. L. &    Conway, J. F. (2005). Virus Maturation: Dynamics and Mechanism of a    Stabilizing Structural Transition that Leads to Infectivity. Current    Opinion in Structural Biology 15, 227-236.-   25. Black, L. W. (1989). DNA Packaging in dsDNA Bacteriophages.    Annual Review of Microbiology 43, 267-292.-   26. Duda, R. L., Hempel, J., Michel, H., Shabanowitz, J., Hunt. D. &    Hendrix, R. W. (1995). Structural Transitions During Bacteriophage    HK97 Head Assembly. Journal of Molecular Biology 247, 618-635.-   27. Galisteo, M. L. & King, J. (1993). Conformational    Transformations in the Protein Lattice of Phage P22 Procapsids.    Biophysical Journal 65, 227-235.-   28. Kunzler, P. & Hohn, T. (1978). Stages of Bacteriophage Lambda    Head Morphogenesis: Physical Analysis of Particles in Solution.    Journal of Molecular Biology 122, 191-211.-   29. Imber, R., Tsugita, A., Wurtz, M. & Hohn, T. (1980). Outer    Surface Protein of Bacteriophage Lambda. Journal of Molecular    Biology 139, 277-295.-   30. Newcomb, W. W., Homa, F. L., Thomsen, D. R., Booy, F. P.,    Trus, B. L., Steven, A. C., Spencer, J. V. & Brown, J. C. (1996).    Assembly of the Herpes Simplex Virus Capsid: Characterization of    Intermediates Observed During Cell-free Capsid Formation. Journal of    Molecular Biology 263, 432-446.-   31. Conway, J. F., Duda, R. L., Cheng, N., Hendrix, R. W. &    Steven, A. C. (1995). Proteolytic and Conformational Control of    Virus Capsid Maturation: The Bacteriophage HK97 System. Journal of    Molecular Biology 253, 86-99.-   32. Santoro, M. M. & Bolen, D. W. (1988). Unfolding Free Energy    Changes Determined by the Linear Extrapolation Method. 1. Unfolding    of Phenylmethanesulfonyl Alpha-Chymotrypsin Using Different    Denaturants. Biochemistry, 18, 8063-8068.-   33. Pace, C. N. & Shaw, K. L. (2000). Linear Extrapolation Method of    Analyzing Solvent Denaturation Curves. Proteins Suppl. 4, 1-7.-   34. Santoro, M. M. & Bolen. D. W. (1992). A Test of the Linear    Extrapolation of Unfolding Free Energy Changes over an Extended    Denaturant Concentration Range. Biochemistry, 31, 4901-4907.-   35. Bolen, D. W. & Santoro. M. M. (1988). Unfolding free energy    changes determined by the linear extrapolation method. 2.    Incorporation of delta G degrees N-U values in a thermodynamic    cycle. Biochemistry 18, 8069-8074.-   36. Yang, Q. & Catalano, C. E. (2003). Biochemical characterization    of bacteriophage lambda genome packaging in vitro. Virology, 305,    276-87.-   37. Gaussier, H., Yang, Q. & Catalano, C. E. (2006). Building a    virus from scratch: assembly of an infectious virus using purified    components in a rigorously defined biochemical assay system. J Mol    Biol 357, 1154-66.-   38. Murialdo, H. & Becker, A. (1978). Head Morphogenesis of Complex    Double-Stranded Deoxyribonucleic Acid Bacteriophages.    Microbiological Reviews 42, 529-576.-   39. Medina, E. (2010). Growing Pains of Bacteriophage Lambda:    Examination of the Maturation of Procapsids into Capsids. Ph.D.,    University of Washington.-   40. Yang, Q., Maluf, N. K. & Catalano, C. E. (2008). Packaging of a    Unit-Length Viral Genome: The Role of Nucleotides and the gpD    Decoration Protein in Stable Nucleocapsid Assembly in Bacteriophage    Lambda. Journal of Molecular Biology 383, 1037-1048.-   41. Lander, G. C., Evilevitch, A., Jeembaeva, M., Potter, C. S.,    Carragher, B. & Johnson, J. E. (2008). Bacteriophage Lambda    Stabilization by Auxiliary Protein gpD: Timing, Location, and    Mechanism of Attachment Determined by Cryo-EM. Structure 16,    1399-1406.-   42. Conway, J. F., Cheng, N., Ross. P. D., Hendrix. R. W.,    Duda, R. L. & Steven. A. C. (2007). A Thermally Induced Phase    Transition in a Viral Capsid Transforms the Hexamers, Leaving the    Pentamers Unchanged. Journal of Structural Biology 158, 224-232.-   43. Jardine, P. J. & Coombs, D. H. (1998). Capsid Expansion Follows    the Initiation of DNA Packaging in Bacteriophage T4. Journal of    Molecular Biology 284, 661-672.-   44. Lee, K. K., Tsuruta, H., Hendrix, R. W., Duda. R. L. &    Johnson, J. E. (2005). Cooperative Reorganization of a 420 Subunit    Virus Capsid. Journal of Molecular Biology 352, 723-735.-   45. Lata, R., Conway, J. F., Cheng, N., Duda, R. L., Hendrix, R. W.,    Wikoff, W. R., Johnson, J. E., Tsuruta, H. & Steven, A. C. (2000).    Maturation Dynamics of a Viral Capsid: Visualization of Transitional    Intermediate States. Cell 100, 253-263.-   46. Fuller. D. N., Raymer, D. M., Kottadiel, V. I., Rao, V. B. &    Smith, D. E. (2007). Single phage T4 DNA packaging motors exhibit    large force generation, high velocity, and dynamic variability. Proc    Natl Acad Sci USA 104, 16868-73.-   47. Tsay, J. M., Sippy, J., Feiss, M. & Smith, D. E. (2009). The Q    motif of a viral packaging motor governs its force generation and    communicates ATP recognition to DNA interaction. Proc Natl Acad Sci    USA 106, 14355-60.-   48. Salunke, D. M., Caspar. D. L. & Garcea, R. L. (1989).    Polymorphism in the Assembly of Polyomavirus Capsid Protein VP1.    Biophysical Journal 56, 887-900.-   49. Salunke, D. M., Caspar, D. L. D. & Garcea, R. L. (1986).    Self-assembly of purified polyomavirus capsid protein VP1. Cell 46,    895-904.-   50. Brady. J. N., Winston, V. D. & Consigli, R. A. (1977).    Dissociation of Polyoma Virus by the Chelation of Calcium Ions Found    Associated with Purified Virions. The Journal of Virology 23,    717-724.-   51. Yanagi, K. & Harada, S. (1989). Destabilization of Herpes    Simplex Virus Type 1 Virions by Local Anesthetics, Alkaline pH, and    Calcium Depletion. Archives Virology 108, 151-159.-   52. Sternberg, N. & Weisberg, R. (1977). Packaging of Coliphage    Lambda DNA: II. The Role of the Gene D Protein. Journal of Molecular    Biology 117, 733-759.-   53. Sternberg, N. & Hoess, R. H. (1995). Display of peptides and    proteins on the surface of bacteriophage lambda. Proceedings of the    National Academy of Sciences 92, 1609-1613.-   54. Bode, V. C. & Harrison, D. P. (1973). Distinct Effects of    Diamines, Polyamines, and Magnesium ions on the Stability of Lambda    Phage Heads. Biochemistry 12, 3193-3196.-   55. Gertsman, I., Gan, L., Guttman, M., Lee, K., Speir, J. A.,    Duda. R. L., Hendrix. R. W., Komives, E. A. & Johnson, J. E. (2009).    An Unexpected Twist in Viral Capsid Maturation. Nature 458, 646-650.-   56. Nurmemmedov, E., Castelnovo, M., Medina, E., Catalano, C. E. &    Evilevitch, A. (2012). Challenging Packaging Limits and Infectivity    of Phage Lambda. Journal of Molecular Biology 415, 263-273.-   57. Fuller, D. N., Rickgauer, J. P., Jardine, P. J., Grimes, S.,    Anderson, D. L. & Smith, D. E. (2007). Ionic effects on Viral DNA    Packaging and Portal Motor Function in Bacteriophage phi29.    Proceedings of the National Academy of Sciences 104, 11245-11250.-   58. Yang. Q., Catalano, C. E. & Maluf, N. K. (2009). Kinetic    Analysis of the Genome Packaging Reaction in Bacteriophage Lambda.    Biochemistry 48, 10705-10715.-   59. Baldwin, R. L. (1986). Temperature Dependence of the Hydrophobic    Interaction in Protein Folding. Proc Natl Acad Sci USA 83,    8069-8072.-   60. Makhatadze, G. I. & Privalov, P. L. (1992). Protein Interactions    with Urea and Guanidinium Chloride: A Calorimetric Study. Journal of    Molecular Biology 226, 491-505.-   61. Schellman, J. A. (1997). Temperature, Stability, and the    Hydrophobic Interaction. Biophysical J. 73, 2960-2964.-   62. Myers, J. K., Pace, C. N. & Scholtz, J. M. (1995). Denaturant m    values and Heat Capacity Changes: Relation to Changes in Accessible    Surface Areas of Protein Unfolding. Protein Science 4, 2138-2148.-   63. Tzlil, S., Kindt, J. T., Gelbart. W. M. & Ben-Shaul, A. (2003).    Forces and Pressures in DNA Packaging and Release from Viral    Capsids. Biophysical Journal 84, 1616-1627.-   64. Evilevitch, A., Lavelle, L., Knobler, C. M., Raspaud, E. &    Gelbart. W. M. (2003). Osmotic Pressure Inhibition of DNA Ejection    from Phage. Proceedings of the National Academy of Sciences 100,    9292-9295.-   65. Nurmemmedov, E., Castelnovo, M., Catalano, C. E. &    Evilevitch, A. (2007). Biophysics of viral infectivity: matching    genome length with capsid size. Q Rev Biophys 40, 327-56.-   66. Yang, F., Forrer, P., Dauter, Z., Conway, J. F., Cheng, N.,    Cerritelli, M. E., Steven, A. C., Pluckthun, A. & Wlodawer, A.    (2000). Novel Fold and Capsid-Binding Properties of the Lambda Phage    Display Platform Protein gpD. Nature Structural Biology 7, 230-237.-   67. Iwai, H., Forrer, P., Pluckthun, A. & Guntert, P. (2005). NMR    Solution Structure of the Monomeric Form of the Bacteriophage Lambda    Capsid Stabilizing Protein gpD. Journal of Biomolecular NMR 31,    351-356.-   68. Rao. V. B. & Black, L. W. (1985). DNA packaging of bacteriophage    T4 proheads in vitro. Evidence that prohead expansion is not coupled    to DNA packaging. J Mol Biol 185, 565-78.-   69. Zhang. Z., Kottadiel, V. I., Vafabakhsh, R., Dai, L., Chemla, Y.    R., Ha, T. & Rao, V. B. (2011). A Promiscuous DNA Packaging Machine    from Bacteriophage T4. PLoS Biol 9, e1000592.-   70. Tomka, M. A. & Catalano, C. E. (1993). Physical and kinetic    characterization of the DNA packaging enzyme from bacteriophage    lambda. J Biol Chem 268, 3056-65.-   71. Filutowicz, M., Grimek, H. & Appekt, K. (1996). Purification of    the Escherichia coli Integration Host Factor (IHF) in One    Chromatographic Step. Gene 147, 149-150.

EXAMPLE 2

With the development of phage display as a platform for vaccines, viralnanoparticles are transitioning from research tools to therapeuticdelivery systems. The popularity of lambda in phage display applicationshas been due in part to the ease in fusing functional peptides andproteins to the capsid decoration protein gpD. The full potential of thelambda capsid as a therapeutic nanoparticle has yet to be exploited andwe are developing bacteriophage lambda for use as a theragnosticnanoparticle system.

To date, all gpD fusion constructs that have been used for phage displayapplications have been constructed in vivo. These systems have thus beenlimited to peptide and protein fusion constructs expressed withinEscherichia coli cells in the context of an infectious virus. Forinstance, a gpD-yellow fluorescence protein fusion construct (gpD-EYFP)expressed from a lambda lysogen in vivo affords infectious virusparticles decorated with EYFP {Alvarez, 2007 #680}. We have previouslydemonstrated that purified gpD can be efficiently added to expandedlambda capsids in a defined biochemical reaction mixture in vitro. Herewe demonstrate that purified gpD-GFP can similarly be used to decoratethe lambda capsid in vitro. We have further constructed severalsingle-cysteine gpD proteins that allow site-specific modification ofthe decoration protein with non-proteinaceous ligands. We show that onesuch construct, gpD(S42C), can be selectively modified with mannose. Theresulting synthetic gpD “glycoprotein” is functional in the capsiddecoration reaction. Finally, we demonstrate that capsids can bemodified with gpD-wild type, gpD-GFP, and gpD-mannose decorationproteins in a defined and tunable surface density. Importantly, thepoly-display shell is competent in the DNA packaging reaction in vitro,indicating that the capsid can be filled with functional genes asdesired. This work sets the stage for the construction of “designer”nanoparticles that can be manufactured to display peptides. proteins,carbohydrates, synthetic polymers, and small molecules in a definedcomposition, symmetrically displayed on the shell surface, and that cancarry specific genes for targeted delivery.

We have developed a hybrid theragnostic nanoparticle assembly thatharnesses the bacteriophage lambda system for the targeted delivery ofdrugs and molecular probes. This invention is based on the assembly ofengineered viral capsid shells in vitro using purified scaffold andmajor capsid proteins. For delivery applications. DNA is efficientlypackaged into the decorated capsids and can modified to carry specificgenes of interest. In addition, the surface of the particle issymmetrically decorated with an external capsid protein that assemblesas trimer spikes at the 140 three-fold icosahedral axes. The decorationprotein can be specifically conjugated with protein and/or syntheticmoieties in defined ratios to enhance cellular targeting/uptake of theparticle, to avoid immune surveillance, or alternatively, to enhanceimmune response to the capsid as a defined antigenic particle. Theseengineered viral nanoparticles can be tailored in specific ways toafford a delivery vehicle with defined surface characteristics for bothdiagnostic and therapeutic applications.

Introduction

A brief background of lambda replication relevant to developing a lambdananoparticle is presented in FIG. 7A. The capsid assembly pathway invivo initiates with the assembly of the viral portal protein (gpB) intoa dodecameric ring-like structure. The gpNu3 scaffolding proteinmediates ring assembly and then chaperones self-assembly of the themajor capsid protein (gpE) into an icosahedral shell. This “immatureprocapsid” contains 420 copies of gpE, a single portal ring situated ata unique vertex, and ˜200 copies of gpNu3 inside the shell. A viralprotease (gpC) is also incorporated into the immature capsid, whichtrims 12 residues from the N-terminus of gpB, digests gpNu3 andauto-proteolyzes so that the internal proteins can exit the shell. Thisaffords the “mature procapsid” into which viral DNA is packaged.

Genome packaging is catalyzed by a terminase enzyme. Upon packaging ca.15 kb duplex DNA, the shell undergoes a remarkable expansion processwhich results in thinning and increased angularization of the shell anda 2-fold increase in capsid volume. The gpD “decoration” protein isexpressed in high-concentration in the phage-infected cell and is amonomer in solution; however, the protein self-assembles as trimerspikes at the 140 three-fold icosahedral axes of the expanded shell (420copies per shell). GpD stabilizes the shell such that it can withstandthe tremendous force generated upon packaging the 48.5 kb genome (>25atmospheres). Subsequent addition of finishing proteins and a viral tailafford an infectious lambda particle (FIG. 7A).

The in vitro assembly system described in Example 1 provides anopportunity to decorate the lambda capsid with gpD-modified constructsunder defined reaction conditions. Here we describe procedures thatallow the assembly of hybrid theragnostic nanoparticles that harnessesthe bacteriophage lambda system.

Lambda capsid assembly in vivo initiates with the formation of theportal complex, a dodecameric ring through which DNA enters the capsidduring packaging and exits during infection of the host.Co-polymerization of the scaffolding protein (gpNu3) and the majorcapsid protein (gpE) nucleates at the portal, forming an immatureprocapsid shell composed of 415 copies of gpE (FIG. 7A). Within theprocapsid interior are ˜70-200 copies of gpNu3 and 10 copies of theprotease (gpC). Residing at one of the 5-fold vertices of theicosahedral shell is the portal. The immature icoahedral shell isrounded in appearance with a diameter of 50-55 nm. Proteolysis by gpCcauses maturation of the procapsid with the removal of gpC and gpNu3from procapsid interior. Partial cleavage of the portal structure whereapproximately half of the portal proteins (gpB) is degraded to B* withthe cleavage of the 20 N-terminal residues also occurs during procapsidmaturation. The terminase complex packages lamdba DNA into the capsidinterior, triggering expansion of the procapsid to the more angularcapsid when approximately 30% is packaged. During expansion gpEundergoes conformational changes, which forms openings in theicosahedral capsid at its 3-fold vertices through which DNA can escape.To stabilize the expanded capsid, 420 copies of the head protein (gpD)add as trimers to the 3-fold vertices of the icosahedral capsid.

Lambda capsid expansion is strictly required for gpD addition. In vivoexpansion is triggered during DNA packaging. In vitro expansion can betriggered by DNA packaging; however, it can also be artificiallytriggered by the denaturant urea, as described in example 1. Theexpanded capsids are then decorated with gpD in vitro, rendering themstable and biologically functional. Furthermore, tails can be attachedto mature capsids forming biologically viable lambda viruses.

Phage display exploits gpD two-fold: its symmetric display and high copynumber, at the surface of the icosahedral capsid. However, it iscurrently limited to proteinaceous ligands decorating an infectiousvirus since N- and C-terminal gpD fusion proteins are expressed fromlysogens in vivo. Because gpD was be expressed and purified at highconcentration and purity for structural determination, we constructedgpD expression vectors to show that gpD can be modified in vivo and invitro and that the modified gpD can decorate the capsid surface incombination at defined ratios.

Materials and Methods

Materials.

Terrific broth (Difco), tryptone, yeast extract, ampicillin, urea, andThermoScientific “Halt”® EDTA-free protease inhibitor cocktail (100×)were purchased from Fisher Scientific. Mature λ DNA (cI857ind 1 Sam 7)was purchased from Invitrogen. Restriction enzymes were purchased fromNew England BioLabs. Chromatography media was purchased from GEHealthcare Life Sciences. All other materials were of the highestquality available. Unless otherwise stated, the pH of all buffers wasadjusted at 4° C. Cell lysis utilized a Thermo Scientific IEC “French”laboratory press. All protein purifications utilized the AmershamBiosciences ÄKTApurifier™ core 10 System from GE Healthcare and sucrosegradients were prepared on a BioComp 107ip Gradient Master. Proteinconcentrations were determined spectrally using a ThermoScientificNanoDrop 2000c spectrophotometer.

Construction of pT₇Cap Dam7am43.

The plasmid pT₇capDam7am43 expresses the lambda major capsid protein(gpE). the scaffolding protein (gpNu3), the portal protein (gpB) and thecapsid protease (gpC), which spontaneously assemble functionalprocapsids that can be purified in high yield {Yang, 2003 #16}. Thisvector was constructed by modification of pT₇cap {Yang, 2003 #16} toobviate the expression of gpD; codons 7 (UUU) and 43 (UCC) were mutatedto amber stop codons (UAG) using the QuikChange II site-directedmutagenesis kit (Agilent) according to the manufacturers instructions.

Expression and Purification of Lambda Procapsids.

Expression of procapsid proteins from pT₇capDam7am43 was performed aspreviously described for pT₇cap {Yang 2003 #16} except that Terrificbroth was used in place of 2xYT media. Procapsid were purified asdescribed, with minor modification, and unless otherwise indicated allprocedures were conducted at 4° C. and with ice-cold buffers. Briefly,the cell pellet was resuspended in 50 ml Buffer A (50 mM Tris, pH 8,containing 10 mM MgCl₂, 100 mM NaCl) containing 2 μg/ml DNase I and thecells were lysed by French press (2-3 passages, 650 psi). The lysate wasclarified by centrifugation (7,650×g×25 minutes) and the procapsids werethen harvested by centrifugation (131,453×g×3 hours). The procapsidswere resuspended by overlaying 5 mL Buffer A onto the pellet overnightat 4° C. The supernatant was aspirated and then diluted with 15 mL™buffer (50 mM Tris, pH 8, containing 20 mM MgCl₂), loaded onto a HiTrapQ (5 mL) column equilibrated with TM Buffer, and then eluted with alinear gradient to 1 molar NaCl. The procapsid containing fractions(eluting at ˜100 mM NaCl) were pooled, dialyzed against Buffer A,concentrated in Amicon Ultra-15 Filters (Millipore), applied to a 10-40%sucrose gradient in the same buffer, and centrifuged for 3 hours at27,000 rpm (SW28 rotor). The procapsid band was visualized in ambientlight, harvested by aspiration, and dialyzed against Buffer A. Thedialyzed procapsids were then concentrated in Amicon Ultra-15 Filtersbetween 100 and 300 nM prior to storage at 4° C.

Construction of Variant gpD Expression Plasmids.

A summary of the proteins described in this work is presented in FIG.2B, each of which was expressed from plasmids constructed as follows.First, vectors that express wild-type gpD protein, without and with aN-terminal hexaHistidine tag (gpD-WT and H6-gpD-WT, respectively) wereconstructed by PCR amplification of the D gene using genomic λ DNA asthe template. The primers are described in Table 2 and the plasmids inTable 3. The expected PCR products were purified using the Wizard SV Geland PCR Clean-up System (Promega), digested with NdeI and BamHI, andcloned into similarly digested pET21a (Novagen) to afford plasmids p(D)and p(H6D), both of which served as templates for mutagenesis of serine42 to cysteine 42 via the QuikChange II site-directed mutagenesis kit(Agilent). The subsequent plasmids expressed mutated gpD proteins(gpD(S42C) and H6-gpD(S42C); FIG. 8B).

To generate gpD proteins containing N- and C-terminal linkersterminating with a unique cysteine residue (cys-gpD-H6 and H6-gpD-cys,respectively; FIG. 8B), the D gene was amplified by PCR using genomic λDNA as a template and the primers presented in Table 2. The plasmidswere constructed in the same manner as those described above.

To construct the plasmid expressing the N-terminal 6-histidine-taggedgpD, the D gene with a linker region encoded following its 3′-end wasinserted in-frame into the vector pRSET_EmGFP (Invitrogen) between therestriction sites NdeI and NcoI. The PCR primers are described in Table2. The plasmid was constructed in a similar manner as those describedabove.

Multiple cloning sites were added to the 5′- and 3′-end of the D geneseparately to generate plasmids that will express proteins of choicefused to gpD via linkers at the N- and C-termini, respectively. Theplasmid was constructed in a similar manner as those described above.The PCR primers are described in Table 2.

TABLE 2 Oligonucleotide primers. Primer Sequence MutagenesisDam7 mut for 5′-CGAGCAAAGAAACC TGA ACCCATTACC-3′ (SEQ ID NO: 11)Dam7 mut rev 3′-GGTAATGGGT TC AGGTTTCTTTGCTCG-5′ (SEQ ID NO: 12)Dam43 mut for 5′-GCTGATGCTGGACACCT GA AGCCGTAAGCTGGTTGC-3′(SEQ ID NO: 13) Dam43 mut rev 3′-GCAACCAGCTTACGGCT TCAGGTGTCCAGCATCAGC-5′ (SEQ ID NO: 14) D(S42C) mut for5′-CCGCTGATGCTGGACACCTGCAGCCGTAAGCTGGTTGC-3′ (SEQ ID NO: 15)D(S42C) mut rev 3′-GCAACCAGCTTACGGCTGCAGGTGTCCAGCATCAGCGG-5′(SEQ ID NO: 16) Recombination D forward 5′-GTGTAAGGGATG CA TATGACGAGC-3′(SEQ ID NO: 17) D-NdeI-BamHI for 5′-CACACCAGTGTAA CAT ATG GGATCCACGAGCAAAGAAACC-3′ (SEQ ID NO: 16) D rev 3′-GTGATGAAGGG G A TCCTTAAACGATGC-5′ (SEQ ID NO: 19) H6-D for 5′-CGATTTGCTGAACCATATGCACCATCACCACCATCACACGAGCAAAGAAACC -3′ (SEQ ID NO: 20) D-H6 rev3′-GCCGCACAGG GAT CCTTT TAGTGATGATGGTGATGATGAACGATGCTG-5′(SEQ ID NO: 21) cys-D for 5′-GCCGTTAACGAT CATATGTGCGGATCAGGGTCAGGGAGTGGTAGCACGAGCAAAGAAACC-3′ (SEQ ID NO: 22)D-cys rev 3′-CCCGTAAAAA AAGCC T C GAG TTAGCAACTTCCTGATCCAGAGCCAGATCCAACGATGCTGATTGC-5′ (SEQ ID NO: 23) MCS-D for5′-GCTGAACACACCAG CTAGC GGGGGGACTGCGACGAGCAAAGAAACC-3′ (SEQ ID NO: 24)D-MCS rev 3′-GGCGGCCTTTAG GCTA G C ACCTCCAAGTCCAACGATGCTGATTGC-5′(SEQ ID NO: 25) D-GFP rev 3′-CCCGTAAAAA CCATGGCAGTGCCGCCGCTTCCTCCTCCAGAGCCAAGTCCAACGATGCTGATTGCC-5′ (SEQ ID NO: 26)Original DNA sequences are underlined. Restriction sites are indicatedby italics. Stop codons are in bold.

TABLE 3 Plasmid Parental vector Forward primer Reverse primer For gpDexpression pD pET21a D for D rev pD(S42C) pET21a D(S42C) mut for D(S42C)mut rev pcys-D pET21a cys-D forw D rev pD-cys pET21a D-Ndel-BamHI forwD-cys rev pMCS-D pET21a MCS-D forw D rev pD-MCS pET21a D forw D-MCS revpH6-gpD pET21a H6-D forw D rev pH6-gpD(S42C) pET21a D(S42C) mut forwD(S42C) mut rev pcys-gpD-H6 pET21a cys-D forw D-H6 rev pH6-gpD-cyspET21a H6-D forw D-cys rev pH6-gpD-DGFP pRSET_EmGFP H6-D forw D-GFP revFor procapsid expression pT7capDam7 pKKT7E Dam7 mut forw Dam7 mut revpT7capDam7am43 pKKT7E Dam43 mut forw Dam43 mut rev

Purification of the Modified gpD Proteins.

Expression of the gpD protein constructs was performed as described forprocapsid expression above. For all constructs except gpD-GFP (seebelow), the induced cells were harvested by centrifugation (6,430×g×30minutes) and the cell pellet was resuspended in Buffer B (20 mM Tris, pH8, 20 mM NaCl, 0.1 mM EDTA, 1 mM DTT) containing 2 μg DNase I. The cellswere lysed by French press (2-3 passages, 650 psi) and the lysate wasclarified by centrifugation (7,650×g×25 minutes). The supernatant washeated to 50° C. for 15 minutes and immediately chilled on ice for 15minutes; insoluble protein was removed by centrifugation (10,000×g×45minutes). The supernatant was dialyzed against Buffer B at 4° C.overnight, clarified by centrifugation (10,000×g×10 min) and then loadedonto a HiTrap Q HP (5 mL) column equilibrated with Buffer B; gpD is notretained by the column, and the flow-through was collected andconcentrated using Amicon Ultra-15 Filters. The concentrated protein wasdialyzed overnight against Buffer B, clarified by centrifugation(10,000×g×10 min), and then loaded onto a Superose 6 10/300 (24 mL)column equilibrated with Buffer B. The gpD-containing fractions werecollected, dialyzed overnight against Buffer B, and then stored at 4° C.until use.

Storage of gpD(S42C) in buffer containing β-ME resulted in adduction ofthe reducing agent to the cysteine residue to afford a mixed disulfidethat precluded chemical modification with maleimide reagents (data notshown). Therefore, gpD(S42C)-containing fractions eluting from theSuperose 6 column above were pooled, dialyzed against Buffer B in theabsence of any reducing agent, and TCEP was added to a finalconcentration of 1 mM prior to storage at 4° C.

Finally, H₆-gpD-GFP was expressed as described above, except that 20 mMglucose was added to the growth media. The cell pellet was resuspendedin Buffer H (20 mM Tris, pH 8, 150 mM NaCl, 0.1 mM EDTA, 7 mM β-ME, 25mM imidazole) and the cells were disrupted by sonication (two 10 secbursts separated by a 10-sec break). DNase 1 (2 μg/ml) and ThermoScientific “Halt” EDTA-free protease inhibitor cocktail (IX) was added,and the resuspension was incubated on ice for 30 minutes. The mixturewas again sonicated (10×10 sec pulses separated by 10 sec breaksin-between), and insoluble material was removed by centrifugation(8.000×g×10 minutes). The clarified lysate was loaded onto a HisTrap FF(5 mL) column equilibrated with Buffer H, and the proteins were elutedwith a 10-column volume gradient to 500 mM imidazole. TheH-gpD-GFP-containing fractions (bright green in color) were dialyzedovernight against Buffer H (minus imidazole), concentrated using anAmicon Ultra-0.5 filter and stored at 4° C. until use.

Synthesis of 6′-Maleimidohexanamido-Polyethyleneglycol Mannoside.

A solution of 2-(2-(2-(Amido)ethyoxy-ethoxy)ethyl-O-α-D-mannoside (20mg, 6.4×10² mmol) in methanol (1 ml) was added to a solution of6-maleimidohcxanoic acid N-hydroxysuccinimide ester (25 mg, 7.7×10⁻²mmol) in methanol (0.5 ml). The reaction mixture was stirred at RT for 2hrs. The desired product was obtained after removal of solvent underreduced pressure following purification by silica column chromatography.The identity of product was confirmed by mass spectrometry and by 1H and13C NMR.

Conjugation and Purification of Mannose-Conjugated gpD(S42C).

The H6-gpD(S42C) stock solution was dialyzed into PBS buffer (1 ml) towhich a solution of maleimide-activated mannose in 50 mM PBS buffer, pH6.6 (800 μL), was added. The reaction mixture was stirred at RT for 48hours and then loaded onto a HiTrap Con A (1 ml) column equilibratedwith 20 mM Tris buffer, pH 7.4 (4° C.), containing 0.5 M NaCl, 1 mMMnCl₂, 1 mM CaCl₂. The protein was eluted over a 0-100% gradient toBuffer X (20 mM Tris buffer, pH 6.4 (4° C.), containing 0.2 Mmethyl-α-D-mannopyranoside and 0.5 M NaCl). ThegpD(S42C::sMannose)-containing fractions were pooled and dialyzedagainst 50 mM Tris buffer, pH 8, containing 150 mM NaCl, concentratedusing an Amicon Ultra-0.5 filter and stored at 4° C. until further use.The expected product was confirmed by mass spectrometry.

Procapsid Expansion.

Expanded capsid shells were prepared as previously described in example1, with modification. Briefly, purified procapsids and a freshlyprepared stock of 6 M urea were mixed to afford a reaction mixture (20μL) containing 40 nM procapsids in 10 mM Tris buffer, pH 8, containing2.5 M urea and 1 mM MgCl₂. The reaction was allowed to proceed for 60min on ice, and the expanded capsids were then buffer exchanged into TMNbuffer (10 mM Tris buffer, pH 8, 1 mM MgCl₂) using an Amicon Ultra-0.5Filter (Millipore).

Decoration of Expanded Capsids with gpD Proteins.

Unless otherwise indicated, purified decoration proteins were added toexpanded capsids to afford a reaction mixture containing 40 nM capsidsand 50 μM decoration protein in TMN buffer. The reaction mixture wasincubated at room temperature for 60 min and the capsids analyzed byagarose gel assay (below). When required, unincorporated decorationproteins were removed prior to analysis either by Superose 6 columnchromatography or by buffer exchange using an Amicon Ultra0-0.5 Filter(Millipore).

Capsid Decoration Assay.

Reaction mixtures were applied to a 1.2% agarose gel, and the capsidsfractionated by electrophoresis at 100 V for 180 minutes. The expandedcapsid and gpD-decorated capsid bands were visualized by Coomassiebrilliant blue stain. Video images of the destained gels were capturedusing an EpiChemi darkroom system with a Hamamatsu camera (UVPBioimaging Systems), and quantitation of the bands was performed usingthe ImageQuant® data analysis package (Molecular Dynamics)

Electron Microscopy.

Samples were applied to 300 mesh carbon coated copper grids (ElectronMicroscopy Sciences) treated by negative glow discharge prior to sampleapplication. Staining was achieved using Nano-W (Nanoprobes) accordingto manufacturer protocols. Bright field transmission electron microscopywas performed at the NanoTech User Facility at the University ofWashington on a FEI Tecnai G2 F20 S-Twin TEM.

Genome Packaging Assay.

The genome packaging assay was performed as described in example 1, withmodification. Briefly, decorated and expanded capsids at a finalconcentration of 15 nM were added to a reaction mixture composed of 50mM Tris buffer, pH 8, containing 1.5 mM NaCl, 10 mM MgCl₂, 2 mMspermidine, 0.42 mM β-ME, 1 mM ATP, 50 nM IHF, and 2 nM full-length λDNA. The packaging reaction (20 μL) was initiated with the addition ofterminase to a final concentration of 100 nM and allowed to incubate for30 minutes at room temperature. DNase I was then added to a finalconcentration of 10 μg/mL and incubated for 5 minutes before thereaction was stopped with the addition of phenol:chloroform (21 μL). Theaqueous layer was removed and loaded onto a 0.8% agarose gel. TheDNase-resistant (packaged) DNA was quantified using video densitometryand ImageQuant (Molecular Dynamics) software package.

Agglutination Assay.

Decorated capsids (1 μM gpD equivalent) were added to 10 mM HEPESbuffered saline solution containing 150 mM NaCl, 1 mM CaCl₂ and 1 mMMgCl₂. Concanavalin A (ConA, 1 μM) was then added to initiate thereaction and agglutination was detected by monitoring the increase inabsorbance (350 nm) at 1-minute intervals. α-D-mannose was added to afinal concentration of 5 mM after 20-minutes incubation time tocompetitively displace the sMannose.

Results

The ability to decorate lambda capsids with modified gpD constructsprovides an attractive approach to develop “designer” nanoparticles ofdefined composition and multi-partite, symmetric presentation. Towardsthis end, we first constructed a plasmid that expresses a HIS-taggedprotein composed of an N-terminal gpD domain fused to a C-terminal greenfluorescent protein domain (gpD-GFP; FIG. 8B). This protein is analogousto the previously described gpD-EYFP fusion protein used to decoratephage particles in vivo (Alvarez et al., 2006). GpD-GFP can be purifiedto homogeneity and in high yield.

Decoration of Lambda Capsids with gpD-Green Fluorescent Protein InVitro.

The decoration of expanded lambda capsids with wild-type gpD (gpD-WT)can be conveniently monitored in vitro using an agarose gel assay asdescribed in example 1. Here we utilize this assay to determine whethergpD-GFP can similarly be added to the expanded shell in vitro. In thiscase, the decorated capsids are visualized in two ways: first byCoomassie blue staining for total protein content (FIG. 9A) and secondby fluorescence imaging for the presence of GFP (FIG. 9B). As previouslydemonstrated, capsids decorated with gpD-WT migrate as a distinct,higher mobility band in the agarose gel. In the presence of increasingamounts of gpD-GFP, progressively slower migrating species appear,consistent with decoration with increasing density of gpD-GFP (FIGS. 9A,9B). At maximal gpD-GFP densities, two distinct bands are apparent inthe gel indicating that two populations are present. Exactly what thesetwo species represent is unclear, but we presume that the upper andlower bands are partially vs. completely decorated capsid shells,respectively.

To confirm that the gpD proteins have in fact added to the capsid shell,the capsids were separated from unreacted material using centrifugalfilters and the protein content of the decorated shells was examined bySDS-PAGE; the data clearly indicate that both proteins stably add to thecapsid (FIG. 9C). Importantly, the surface density of both gpD-WT andgpD-GFP can be tuned by adjusting the ratio of these two proteinsincluded in the reaction mixture. In the presence of gpD-WT alone, theratio of major capsid protein (gpE) to gpD-WT in the decorated particlesis ˜1:1, as anticipated for a fully decorated shell capsid (FIG. 7,Table 4). As the amount of gpD-GFP is increased in the reaction mixture,the density of the modified protein on the decorated particles increasesto an average surface density of ˜77% (FIG. 8C; Table 4). Particledecoration does not appear to be linear, suggesting that gpD-WToutcompetes gpD-GFP for addition to the capsid surface (data not shown).This is not surprising given that the GFP domain (26.9 kDa) addssignificant bulk to the small gpD polypeptide (11.4 kDa), which likelyinterferes with the assembly of the trimeric spikes at the capsidsurface (see FIG. 8A).

TABLE 4 Decoration of Lambda Capsids by gpD Variants Surface DensityVariant (% relative to gpD) gpD-WT 100 gpD(S42C) 90 ± 16gpD(S42C::sMannose) 82 ± 12 H₆-gpD-GFP 77 ± 14

Construction of gpD-Vectors for Diverse Phage Display Applications.

The lambda gpD protein has been used in a variety of phage displayapplications {Beghetto, 2011 #688}. To date, the gpD fusion constructshave appended peptides to either the N-terminus or C-terminus of theprotein and decorated phages have been assembled in vivo from theinduction of the modified lambda lysogens. While useful, this approachhas several limitations. First, display in vivo restricts potentialligands to those that do not affect the development of viable viralparticles. It is quite likely that many desired ligands will in factadversely affect the yield of infectious virus. Second, the currentphage display systems require construction of modified lambda genomesand induction of lambda lysogens in vivo to afford the modifiedparticle. This approach is laborious and is ultimately controlled by thewhims of the E. coli cell, which does not allow facile manipulationshell decoration density. We reasoned that the in vitro gpD-decorationprocedure described above could be adapted for the display of anypeptide/protein on the capsid surface and at defined surface densities.Towards this end we constructed a vector that possesses a multi-cloningsite immediately downstream from the D gene (gpD-MCS, FIG. 8B), whichallows facile expression of C-terminal tagged gpD-fusion proteins thatcan be used to decorate the lambda capsid in vitro, as described forgpD-GFP above. We also constructed an analogous vector that allowsfacile generation of N-terminal tagged fusion proteins (MCS-gpD). Thesevectors allow rapid and efficient expression of gpD-fusion proteins thatcan be used to decorate the lambda capsid in vitro with total control ofsurface display density.

A third major limitation to all current phage display platformsincluding lambda is that the systems are limited to proteinaceous tags.To address this limitation, we next constructed vectors that express gpDcontaining a unique cysteine residue either at the N-terminus(Cys-gpD-H₆) or the C-terminus (H₆-gpD-Cys) of the protein (FIG. 8B).The purified proteins provide scaffolds that can be site-specificallymodified with a variety of ligands, not limited to peptides, usingsimple maleimide chemistry. This is discussed further below.

A final limitation of current phage display systems centers on thelocation of the N- and C-termini of gpD bound to the capsid surface. GpDis a monomer in solution but adds to the 140 three-fold axes of theicosahedral shell as a trimeric spike (FIGS. 7, 8A). The N-terminus ofeach subunit directly interacts with the capsid shell to providestabilizing contacts required for shell integrity {Lander, 2008 #351},while the C-termini exit the gpD trimer spike proximate to the shellsurface (FIG. 8A) {Lander, 2008 #351}. In both cases, appending bulkyligands could easily hinder gpD trimer assembly and interfere with itsability to stabilize the DNA-filled shell. Indeed, this was observedwith phage decorated with various GFP variants in vivo {Alvarez, 2007#680; Zeng, 2011 #681; Nicastro et al., 2013} and is consistent with theincomplete decoration observed with gpD-GFP in vitro (see FIG. 9A).

The crystal structure of the gpD trimer spike reveals that Ser42 ispositioned at the apex of the spike in all three subunits (FIG. 8A).Modification of this residue would place the desired tag projecting awayfrom the capsid surface and into solution for optimal display, and withminimal insult to gpD spike assembly and shell integrity. We thereforeconstructed vectors (with and without N-terminal histidine tags) thatthat express gpD in which Ser42 has been changed to Cys42 (H₆-gpD(S42C)and gpD(S42C), respectively; FIG. 8B). Importantly, there are no othercysteine residues in the native protein and this provides a unique sitefor reaction with maleimide-based tags.

Chemical Synthesis of gpD(S42C::sMannose).

Cysteine-modified gpD constructs provide proteins that can beefficiently and specifically modified using maleimide chemistry. Todemonstrate the utility of this approach, we synthesized6′-maleimidohexanamido-polyethyleneglycol mannoside (sMannose) asdescribed in Materials and Methods. This was used to chemically modifygpD(S42C) to afford the gpD(S42C::sMannose) glycoprotein, which waspurified to homogeneity.

We next examined capsid decoration by gpD(S42C::sMannose). Asanticipated, gpD-WT adds to the expanded capsids to afford a unique bandof higher mobility in the agarose gel compared to unexpanded(undecorated) procapsids (FIG. 10A). Increasing the fraction ofgpD(S42C::sMannose) included in the reaction mixture results in gradualretardation of capsid migration in the gel, consistent with progressivedecoration by the gpD glycoprotein. To confirm this presumption, thecapsids were separated from unreacted material and the protein contentof the decorated capsids analyzed by SDS-PAGE. Unlike gpD-GFP, thedensity of gpD(S42C::sMannose) on the decorated particles increases in alinear fashion (data not shown) and to a higher average surface densityof ˜82% (FIG. 10B, Table 4).

The Lambda Capsid can be Decorated with Multiple Tags in a DefinedComposition.

As demonstrated above, modified gpD constructs can be used to decoratethe lambda capsid with a large protein (GFP) and with a mannoseglycoprotein. Here we demonstrate that the shell can be decorated withmultiple ligands simultaneously. Lambda capsids were incubated withgpD-WT, gpD(S42C::sMannose), and gpD-GFP alone or in combination. Thereaction mixture was analyzed by agarose gel assay, which clearlydemonstrates that the proteins, alone and in combination, add to thecapsid shell to afford decorated particles with a unique mobility in thegel (FIGS. 11A, 11B). Analysis of the isolated, decorated shells bySDS-PAGE demonstrates that (i) each construct efficiently adds to theshell and that (ii) all three proteins can add to the capsid whensimultaneously present in the reaction mixture (FIG. 11C). Within thiscontext, we note that gpD-WT and the gpD(S42C::sMannose) glycoproteinmigrate closely in SDS-PAGE. In order to confirm the presence of bothproteins on the decorated capsid surface, we utilized an agglutinationassay. In this assay, the presence of mannose is detected byagglutination of the particles by with Concanavalin (ConA). As expected,neither gpD-WT nor gpD-S42C decorated capsids exhibit a positiveagglutination response, while gpD-GFP decorated capsids exhibit a weak(non-specific) agglutination response (FIG. 5D). In contrast, capsidsdecorated with gpD(S42C::sMannose) show a strong response indicating (i)mannose is present on the capsid surface and (ii) it is efficientlydisplayed for interaction with ConA.

Structural Characterization of the Decorated Lambda Capsids.

A major advantage of utilizing viral capsids as display platforms isthat under ideal conditions they can display the tags in a symmetric anddefined manner. In the case of the gpD constructs discussed here, tagsof known composition can be appended to the display protein in asite-specific manner, which can then be presented as trimers at thethree-fold icosahedral axes of the capsid shell. While it is clear thatour modified gpD constructs efficiently bind to the lambda capsid,confirmation that the shells remain intact for optimal display of thetags is important. We therefore utilized electron microscopy to examinethe decorated shells as described in Materials and Methods. Capsidsdecorated with gpD-WT are composed of an intact icosahedral shell withthinned walls and significant angular facets (FIG. 12), as observed withan infectious lambda virus {Dokland, 1993 #414; Lander, 2008 #351}. ThegpD(S42C) decorated capsids are similar in morphology, consistent withthe conservative mutation in the protein and their near-native gelmigration in the agarose gel (FIG. 11A). Capsids decorated with thegpD(S42C::sMannose) glycoprotein similarly display a thin andangularized shell, though perhaps not as faceted as wild-type decoratedshells (FIG. 12). The gpD-GFP decorated shells also possess the thinwall phenotype and similarly appear less faceted than wild type. Inaddition, the shells have an extra density on the exterior shellsurface, which we presume reflects the presence of the large GFP domainappended to the capsid surface (FIG. 12). Decorated capsids isolatedfrom reaction mixtures containing gpD-WT, gpD(S42C::sMannose), andgpD-GFP in a 40:40:20 ratio also possess extra staining density at thecapsid shell, though not as prominent as those fully-decorated withgpD-GFP; this is consistent with the lower surface density of gpD-GFP onthese particles (FIG. 11A).

Functional Characterization of the Decorated Lambda Capsids.

Procapsids serve as receptacles for the viral genome, and gpD isrequired to provide structural integrity to the DNA-filled shell (FIG.7) (Yang, 2008 #). In the absence of gpD, the capsid cannot withstandthe intense pressures generated by the packaged DNA (>50 atmospheres)and the shell fractures, rendering the DNA accessible to DNase digestion{Yang, 2008 #1}. Here we utilize an in vitro DNA packaging assay todetermine if capsids decorated with modified gpD proteins arefunctionally active in capsid shell stabilization.

The genome packaging reaction was performed as described in Materialsand Methods using capsids decorated with either gpD-WT, gpD(S42C),gpD(S42C::sMannose), or gpD-GFP. As shown in FIG. 7A, gpD(S42C) and thegpD(S42C::sMannose) glycoprotein are as efficient as wild-type gpD inthe DNA packaging reaction. In contrast, capsids decorated withH₆-gpD-GFP are seriously compromised when the surface density of theprotein exceeds 50% (FIG. 7B). Thus, while the GFP-modified protein canfully decorate the expanded shell (Table 4), it does not necessarilyimpart the requisite structural integrity required to retain afully-packaged genome. This is consistent with the observation thatinfectious lambda virus decorated with gpD-GFP variants are less stablecompared to wild-type virus in vivo (Alvarez et al., 2006, Nicastro etal., 2013).

Discussion

Decoration of the lambda capsid is a powerful tool in the development ofnanoparticles for theragnostic applications. Its relative ease aids inthe modification of the capsid, allowing the display of tags in adefined and tunable manner. We have developed an in vitro decorationsystem in which lambda capsids are decorated with non-proteinaceousligand conjugated to gpD. Validation of the system was first confirmedusing the modified gpD fusion protein, gpD-GFP.

Lambda capsids can be decorated in vitro with gpD-GFP. Characterizationof the gpD-GFP in vitro decorated capsids is consistent with thegpD-EYFP and gpD-GFP in vivo decorated viruses (Alvarez et al., 2008;Nicastro et al., 2013). The added bulk of GFP affects the stability ofboth the in vive and in viva decorated capsids. Because the C-terminusof gpD lies near the capsid surface (Chang et al., 2004; Lander et al.,2008), gpD-GFP may interfere with the addition of the gpD trimers to thecapsid surface, hence, the two distinct populations of gpD-GFP decoratedcapsids visualized by agarose gel analysis when only gpD-GFP is present.Representation of the gpD-GFP decorated capsids on SDS-PAGE is anaverage of the two populations. The exact coverage for each populationis unknown, but we surmise that one population has a higher surfacedensity than the other. Despite this, gpD-GFP is ineffective instabilizing capsids to the same extent as the other gpD variants. Theadded bulk of GFP to the C-terminus of gpD most likely interferes withthe ability of gpD to stabilize the capsid. The apical gpD residue 42 isan ideal candidate for conjugation.

The advent of gpD(S42C) (and similar gpD functional mutants) and thecapacity of the system to be tunable circumvents the issues of capsidstability. Modification of the apical residue S42C does not decreasecapsid stability to the same extent as modifications to either the N- orC-termini of gpD Addition of gpD(S42C::sMannose) to the capsid, althoughnot as complete as gpD-WT, does provide sufficient capsid stability.This suggests that full coverage of the capsid shell by gpD may not be astrict requirement for packing competency. About 80% coverage appears tobe the minimum required assuming that the modification tag does notinterfere with structural stability of the capsid, as the case withgpD-GFP but not gpD(S42C::sMannose). Added bulk to the structurallycompact gpD-WT at the apical residue 42 may lower efficiency of trimerformation depending on the size of the conjugant. This may be avoidedwith the initial addition of gpD-WT at a partial complement. Althoughhow the gpD variants are participating in trimer formation is unclear,gpD-WT stabilizes the capsid structure by limiting the saturation of themodified gpD variant and its potential hazard of capsid instabilityeither within in each trimer or between trimers.

The range of capsid decoration is limited by the initial concentrationsof the gpD variants thereby allowing tunability. Capsids decorated withgpD-WT and gpD-GFP appear as diffuse bands on agarose gels compared tocapsids decorated with a single variant (FIGS. 9A, 11A), suggesting thatthe gpD-WT is masking the partiality of gpD-GFP. Capsids decorated withgpD-WT and gpD(S42C::sMannose) appear as discrete bands with increasingconcentrations of gpD(S42C::sMannose) (FIG. 10A). Although the sizedisparity between the two variants is minimal by comparison, thestructural difference between gpD-WT and gpD(S42C::sMannose) is at anapical residue, not at a region involved in capsid stability likegpD-GFP. Decoration of the capsid by gpD(S42C::sMannose) is most likelymore homogenous compared to that by gpD-GFP. Capsids decorated with allthree gpD variants appear as a diffuse band on agarose gel (FIG. 11A),implicating gpD-GFP as the factor causing heterogeneity in capsiddecoration. Because gpD-GFP is initially limited, its role in decorationis depressed but not suppressed. Our system's tunability isadvantageous, allowing the inclusion of proteinaceous andnon-proteinaceous ligands that would otherwise be disadvantageous tocapsid stability.

One potential downstream application of the in vitro decorated capsidswould be their involvement in vitro assembled viruses. We havepreviously developed an assay for assembling via lambda viruses in vitro(Gaussier et al., 2006). Genetically modified DNA can be packaged intoin vitro decorated capsids. The attachment of tails to the mature capsidprevents exit of the packaged DNA as well as produces a carrier for DNAwhich can be targeted to a specific cell type, for example, via anon-proteinaceous modification conjugated to gpD. We have utilized ourin vitro capsid assembly system to decorate lambda capsids withproteinaceous and non-proteinaceous modification. The ability to tunethe decoration of lambda capsids in vitro obviates possible capsidinstability of bulky modifications but not limiting what can be added tothe capsid surface. This expands the utility of the capsid displaysystem.

REFERENCES FOR EXAMPLE 2

-   1. Alvarez, L. J., Thomen, P., Makushok, T., and Chatenay, D. (2007)    Propagation of Fluorescent Viruses in Growing Plaques, Biotechnol.    Bioeng. 96, 615-621.-   2. Nicastro, J., Sheldon, K., El-zarkout, F. A., Sokolenko, S.,    Aucoin, M. G., and Slavcev, R. (2013) Construction and Analysis of a    Genetically Tuneable Lytic Phage Display System, App. Microbiol.    Biotechnol.-   3. Yang, Q., and Catalano, C. E. (2003) Biochemical characterization    of bacteriophage lambda genome packaging in vitro, Virology 305,    276-287.-   4. Gaussier, H., Yang, Q., and Catalano, C. E. (2006) Building a    virus from scratch: assembly of an infectious virus using purified    components in a rigorously defined biochemical assay system, J Mol    Biol 357, 1154-1166.-   5. Medina, E., Nakatani, E., Krusc, S., and Catalano, C. E. (2012)    Thermodynamic Characterization of Viral Procapsid Expansion into a    Functional Capsid Shell, J. Mol. Biol. 418, 167-180.-   6. Beghetto, E., and Gargano, N. (2011) Lambda Display: A Powerful    Tool for Antigen Discovery, Molecules 16, 3089-3105.-   7. Lander, G. C., Evilevitch, A., Jeembaeva, M., Potter, C. S.,    Carragher, B., and Johnson, J. E. (2008) Bacteriophage Lambda    Stabilization by Auxiliary Protein gpD: Timing, Location, and    Mechanism of Attachment Determined by Cryo-EM, Structure 16,    1399-1406.-   8. Zeng. L., and Golding, I. (2011) Following Cell-fate in E. coli    After Infection by Phage Lambda, Journal of Visualized Experiments    56, 3363.-   9. Dokland, T., and Murialdo, H. (1993) Structural Transitions    During Maturation of Bacteriophage Lambda Capsids, J. Mol. Biol.    233, 682-694.-   10. Yang, Q., Maluf, N. K., and Catalano, C. E. (2008) Packaging of    a Unit-Length Viral Genome: The Role of Nucleotides and the gpD    Decoration Protein in Stable Nucleocapsid Assembly in Bacteriophage    Lambda, Journal of Molecular Biology 383, 1037-1048.

We claim:
 1. A theragnostic particle enveloped by a viral particleshell, wherein: at least one engineered decoration protein is bound tothe outer surface of the shell, the at least one engineered decorationprotein comprises the amino acid sequence of SEQ ID NO: 4 (wt gpD),wherein at least one solvent-exposed residue of the at least oneengineered decoration protein is mutated to a cysteine or a non-naturalamino acid, and the at least one solvent-exposed residue is selectedfrom the group consisting of Ser29, Ala30, Lys31, Asp41, Thr42, Ser43,Ser44, Arg45, Lys46, Gly52, Thr53, Thr54, Asp55, Asp67, Gln68, Thr69,Ser70, Thr71, Thr72, Glu90, Ala9, Ala92, Ser93, Asp94, Glu95, Thr96,Lys97, Lys98, Arg99, and Thr100 as corresponding to the amino acidsequence of SEQ ID NO:
 4. 2. The particle of claim 1, wherein the shellcomprises at least one decoration competent viral particle shellselected from the group consisting of bacteriophage lambda,bacteriophage T4, bacteriophage L, bacteriophage P22, bacteriophage 21,bacteriophage P4, herpesvirus, and adenovirus.
 3. The particle of claim1, wherein the shell comprises a bacteriophage lambda viral particleshell.
 4. The particle of claim 1, wherein the shell comprises anexpanded viral particle shell.
 5. The particle of claim 1, wherein theat least one mutated residue is independently conjugated with one ormore compounds independently selected from the group consisting of aproteinaceous agent and a non-proteinaceous agent.
 6. The particle ofclaim 4, wherein the one or more compounds do not naturally occur a in awild type viral capsid.
 7. The particle of claim 4, wherein the one ormore compounds are present on the particle in a defined ratio relativeto the engineered decoration protein.
 8. The particle of claim 4,wherein the one or more compounds comprise two or more differentcompounds, which are present on the particle in a defined ratio relativeto each other.
 9. The particle of claim 4, wherein the non-proteinaceousagent is selected from the group consisting of nucleic acids, lipids,carbohydrates, polypeptides, polymers, organic molecules, inorganicmolecules, and any combinations thereof.
 10. The particle of claim 4,wherein the non-proteinaceous agent is selected from the groupconsisting of therapeutic compounds, diagnostic compounds, adjuvants,antigens, antibodies, and any combinations thereof.
 11. A pharmaceuticalcomposition comprising the theragnostic particle of claim 1 and apharmaceutically acceptable carrier.
 12. An isolated recombinant proteincomprising the amino acid sequence of SEQ ID NO: 4 (wt gpD), wherein atleast one residue of the protein is mutated to a cysteine or anon-natural amino acid, wherein the at least one residue is selectedfrom the group consisting of Ser29, Ala30, Lys1, Asp41, Thr42, Ser43,Ser44, Arg45, Lys46, Gly52, Thr53, Thr54, Asp55, Asp67, Gln68, Thr69,Ser70, Thr71, Thr72, Glu90, Ala91, Ala92, Ser93, Asp94, Glu95, Thr96,Lys97, Lys98, Arg99, and Thr100 as corresponding to the amino acidsequence of SEQ ID NO:
 4. 13. The protein of claim 12, wherein the atleast one mutated residue is independently conjugated with one or morecompounds independently selected from the group consisting of aproteinaceous agent and a non-proteinaceous agent.
 14. An isolatednucleic acid encoding the recombinant protein of claim
 12. 15. Arecombinant expression vector comprising the isolated nucleic acid ofclaim
 14. 16. An isolated host cell comprising the recombinantexpression vector of claim
 15. 17. An in vitro method for preparing atheragnostic particle of claim 1, the method comprising decorating adecoration competent viral particle shell in vitro with a defined amountof at least one engineered decoration protein linked to one or morecompounds, wherein the at least one engineered decoration proteinstabilizes the decoration competent viral particle shell to produce atheragnostic particle, wherein the at least one engineered decorationprotein is as recited in claim
 1. 18. The method of claim 17, whereinthe decoration competent viral particle shell is an expanded viralparticle shell, and wherein method comprises contacting the viralparticle with an expansion agent, thus generating the expanded viralparticle shell.
 19. The method of claim 18, wherein the expansion agentis selected from the group consisting of chaotropic agents, pH, heat,and any combinations thereof.
 20. The method of claim 17, wherein thedecoration competent viral particle shell is at least one selected fromthe group consisting of a bacteriophage lambda, bacteriophage T4,bacteriophage L, bacteriophage P22, bacteriophage 21, bacteriophage L,bacteriophage P4, herpesvirus, and adenovirus.